CN1882702A - Synthetic lethal screen using RNA interference - Google Patents

Abstract

The invention provides a method for identifying one or more genes in a cell of a cell type which interact with, e.g., modulate the effect of, an agent, e.g., a drug. For example, an identified gene may confer resistance or sensitivity to a drug, i.e., reduces or enhances the effect of the drug. The invention also provides STK6 and TPX2 as a gene that exhibits synthetic lethal interactions with KSP encoding a kinesin-like motor protein, and methods and compositions for treatment of diseases, e.g., cancers, by modulating the expression of STK6 or TPX2 gene and/or the activity of STK6 or TPX2 gene product. The invention also provides genes involved in cellular response to DNA damage, and their therapeutic uses.

Description

Disturb the collaborative screening of carrying out that causes death with RNA

The application requires the U.S. Provisional Patent Application No.60/554 of submission on March 17th, 2004 according to 35 U.S.C.119 (e), 284, the U.S. Provisional Patent Application No.60/548 that submitted on February 27th, 2004,568, with the U.S. Provisional Patent Application No.60/505 that submitted on September 22nd, 2003,229 rights and interests, described application is incorporated herein by reference in full at this.

1. invention field

The present invention relates to disturb the screening that interacts, for example, cause death/collaborative (synthetic lethal) method for screening and the composition that cause death with RNA.The invention still further relates to and KSP, promptly kinesin sample dynein has the collaborative interactional gene that causes death, and therepic use.The invention still further relates to the gene of participation to DNA destructive cell response, and therepic use.

2. background of invention

It is the genetic expression that is used for preventing mammalian cell that RNA disturbs (RNAi), and produces effective ways (Couzin, 2002, the Science 298:2296-2297 of a lot of vibrations in scientific domain; McManus et al., 2002, Nat.Rev.Genet.3,737-747; Hannon, G.J., 2002, Nature 418,244-251; Paddison et al., 2002, Cancer Cell 2,17-23).RNA disturbs and guards in the evolutionary process from the caenorhabditis elegant to people, and is considered to not be subjected to work in the RNA viruses intrusion at the protection cell.When cell was subjected to the dsRNA virus infection, it is fixed that dsRNA is identified with target, so that cut by the III type RNA enzyme that is called " nickase (Dicer) ".The nickase enzyme becomes the duplex of short 21nt with RNA " stripping and slicing ", is called siRNA or short RNA interfering, and it comprises the complete paired ribonucleotide of 19nt and two unpaired Nucleotide on every chain 3 ' end.These short duplexs associate with the polyprotein mixture that is called RISC, and these mixtures are navigated to the mRNA transcript that has sequence similarity with siRNA.Therefore, be present in the nuclease cutting mRNA transcript in the RISC mixture, thus the expression of cancellation gene product.Under the situation of virus infection, this mechanism will cause the destruction of virus transcription thing, stop virus synthetic thus.Because siRNAs is double-stranded, the potential that arbitrary chain all has with RISC associates and guidance has the transcript silence of sequence similarity.

The specific gene silence provides utilizes human genome data interpretation gene function, identifies the potential of the treatment that medicine target and exploitation are more special.A lot of such siRNA specificitys that height is provided as their target of target that are applied as.With the hybridization that contains with the transcript of the partial identity of siRNA sequence, it is exophytic not as the phenotype of the silence of the transcript of target to induce reflection to remove target gene.This can make identifies that the gene with phenotypic correlation becomes chaotic.Many reports in the document have been pointed out the accurate specificity of siRNA, pointed out to the requirement of identity (Elbashir etal., the 2001.EMBOJ.20:6877-6888 completely that be close to of siRNA sequence; Tuschl et al., 1999, Genes Dev.13:3191-3197; Hutvagneret al., Sciencexpress 297:2056-2060).A up-to-date report shows, the sequence complementarity is that the fixed transcript cutting of siRNA target is necessary completely, to under the situation of not carrying out the transcript degraded, the mode with microRNA cause translation repression (Hutvagner et al., Sciencexpress 297:2056-2060) and part is complementary.

To little adjusting RNA, comprise that the function of siRNA and miRNA does not also fully understand.A ubiquitous problem relates to the mechanism of the different reticent approach of determining this two classes adjusting RNA.MiRNA is the adjusting RNA by genomic expression, and from the processing of precursor stem-ring structure, is incorporated into single-chain nucleic acid (Leeet al., 1993, the Cell 75:843-854 of the sequence among 3 ' UTR of said target mrna with generation; Reinhart et al., 2000, Nature 403:901-906; Lee et al., 2001, Science 294:862-864; Lau et al., 2001, Science 294:858-862; Hutvagner et al., 2001, Science 293:834-838).MiRNA combines (Zeng et al. with the transcript sequence that only has the part complementarity, 2002, Molec.Cell 9:1327-1333), and under the situation that does not influence the steady state RNA level, prevent translation (Lee et al., 1993, Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862).MiRNA and siRNA be by " cutting unit " processing, and with the composition of the reticent mixture of RNA inductive associate (Hutvagner et al., 2001, Science293:834-838; Grishok et al., 2001, Cell 106:23-34; Ketting et al., 2001, Genes Dev.15:2654-2659; Williams et al., 2002, Proc.Nntl.Acad.Sci.USA 99:6889-6894; Hammond et al., 2001, Science 293:1146-1150; Mourlatos et al., 2002, Genes Dev.16:720-728).A nearest report (Hutvagner et al., 2002, Sciencexpress 297:2056-2060) has been supposed only by determining by the adjusting of miRNA approach to the siRNA approach with the complementary degree of target transcript.Only infer that to have a siRNA of partial identity similar to miRNA to the mRNA target, will in translation repression, work, rather than cause RNA and degrade.

Also verified, siRNA and shRNA can be used for making in vivo gene silencing.Utilize siRNA and shRNA to carry out the ability of gene silencing in vivo, have the potential that makes it possible to select and develop siRNA for therepic use.Nearest report has been emphasized the potential treatment application of siRNA.The apoptosis of Fas mediation keeps liver with the hepatopathy spectrum is relevant widely and can pass through to suppress hepatocellular apoptosis death.(Song et al.2003, Nat.Medicine 9,347-351) due to the siRNA of Fas acceptor mouse carried out intravenous injection with target for Song.In mouse liver cell, at mRNA and protein level the Fas gene is carried out silence, stop apoptosis, and the protection mouse avoids hepatitis inductive liver destruction.Like this, reticent Fas expresses, and has kept avoiding the treatment prospect that cytotoxicity prevents liver injury by the protection liver cell.As another example, the siRNA that decides TNF-α with target carries out peritoneal injection to mouse.Suppress lipopolysaccharide-induced TNF-α genetic expression, and protected these mouse to avoid Sepsis.The concentrated area, these results suggest siRNA can work in vivo, and have potential as medicine (Sorensen et al., 2003, J.Mol.Biol.327,761-766).

People such as Martinez have reported and can disturb the selectivity target to decide oncogene mutation (Martinez et al., 2002, Proc.Natl.Acad.Sci.USA 99:14849-14854) with RNA.In this report, proved that the siRNA in zone of R248W mutant that target contains the p53 of point mutation surely makes the expression silencing of sudden change p53, but do not made the expression silencing of wild type p53.

Wilda etc. have reported that the siRNA that target can be decided the M-BCR/ABL fusion mRNA is used for removing leukemia cell's M-BCR/ABL mRNA and M-BRC/ABL cancer protein (Wilda et al., 2002, Oncogene 21:5716-5724).But this report also shows siRNA and Imatinib (a kind of small molecules ABL kinases tyrosine kinase inhibitors) united and is used for the leukemia cell, does not further increase apoptosis induced.

U.S. Patent No. 6,506,559 disclose the RNA interference method of the expression of target gene that is used for suppressing cell.This method comprise with have at double stranded region with target gene in the partially or completely double-stranded RNA transfered cell of the sequence that is equal to of sequence in or in the transfered cell external environment.RNA sequence with insertion, disappearance and simple point mutation with respect to target sequence also is found and can effectively suppresses to express.

It is the RNA interference of RNA fragment in the fruit bat vitro system of 21-23 Nucleotide (nt) that U.S. Patent Application Publication No.US2002/0086356 discloses employing length.This patent application has openly been instructed when with these 21-23nt fragment purifications and when being added back in the fruit bat extract, and the sequence-specific RNA when there is not long dsRNA in their mediations disturbs.This patent application has openly also been instructed, and also the oligonucleotide with chemosynthesis of same or similar character can be used for target and decide specific mrna, is used for degrading at mammalian cell.

It is that the double-stranded RNA (dsRNA) of 19-23nt is induced the sequence specific post transcriptional gene silencing in the fruit bat vitro system that PCT open WO02/44321 discloses length.This PCT has openly instructed by III type RNA enzyme sample processing reaction from the short interfering rna (siRNA) of long dsRNA generation or the siRNA duplex with 3 ' outstanding end of chemosynthesis, can mediate the effective target RNA cutting in the lysate, and cleavage site is positioned near the center in the guiding zone that siRNA crossed over.This PCT openly also provides evidence, proves that the direction of dsRNA processing has been determined to be cut with still antisense target RNA of justice by the siRNP mixture that produces.

U.S. Patent Application Publication No.US 2002/016216 discloses the method that weakens expression of target gene in the cultured cells, this be by with double-stranded RNA (dsRNA) realizing in the amount transfered cell that is enough to weaken expression of target gene, described double-stranded RNA is included under the stringent condition nucleotide sequence with the nucleotide sequence hybridization of target gene.

The open WO 03/006477 of PCT discloses the RNA precursor of through engineering approaches, when it is expressed in cell, process by cell, to produce the fixed siRNA (siRNA) of target, described siRNA disturbs (RNAi) approach optionally to make the fixed gene silencing of target (by cleavage specificity mRNA) with the RNA of cell self.This PCT has openly instructed in the nucleic acid molecule transfered cell by the RNA precursor of these through engineering approaches of will encoding with proper regulation sequence, can be on time and space, that is, in specified time and/or the optionally expression of the RNA precursor of control engineeringization in particular organization, organ or cell.

To the discussion of reference with quote, should not be interpreted as admitting that described reference is a prior art of the present invention herein.

3. summary of the invention

The invention provides and use RNA to disturb identified gene or its product and a kind of reagent, as the interaction between medicine and/or another kind of gene or its product, as causing death/the collaborative interactional method and composition that causes death.The present invention also provides collaborative the causing death of utilizing between STK6 kinases or TPX2 and the kinesin sample dynein KSP inhibitor to interact and treatment method for cancer and composition.The present invention also provides the gene that participates in DNA destructive cell response, and therepic use.

On the one hand, the invention provides the method for identified gene, the product of described gene is regulated the effect of a kind of reagent to a kind of cell of cell type.This method comprises that (a) makes a large amount of group of one or more cells of described cell type contact with described reagent, wherein the group of each described one or more cell comprises one or more the different siRNAs (siRNA) from a large amount of different siRNA, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target; (b) effect to the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; If (c) effect of the cell of the described cell type of arbitrary described heterogeneic siRNA is different calmly to not comprising target to the effect of the group of described one or more cells of described one or more different siRNA of comprising the fixed described gene of target and described reagent for described reagent, then identified gene is that described its product is regulated the gene of described reagent to the effect of the cell of described cell type.In one embodiment, comprise one or more each described groups of cells among described a large amount of siRNA by before described contact procedure, obtaining with described one or more siRNA transfections.In one embodiment, the component to each described one or more cell drives capable contact procedure (a) into.

In a kind of specific embodiment, the invention provides the method for identified gene, the product of described gene is regulated the effect of a kind of reagent to a kind of cell of cell type, described method comprises that (a) is with comprising from a large amount of group of one or more cells of the described cell type of composition transfection of one or more different siRNAs (siRNA) of a large amount of different siRNA each, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target; (b) described a large amount of group of one or more cells is contacted with described reagent; (c) effect to the cell of the described cell type of the fixed arbitrary described heterogeneic siRNA transfection of target of no use compares to the effect of the group of each described one or more cell and described reagent with described reagent; If (d) effect of the cell of the described cell type of arbitrary described heterogeneic siRNA is different calmly to not comprising target to the effect of the group of described one or more cells of described one or more different siRNA of comprising the fixed described gene of target and described reagent for described reagent, then identified gene is that described its product is regulated the gene of described reagent to the effect of the cell of described cell type.

With described reagent the effect of the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target is compared, described reagent can strengthen the effect of the group of each described one or more cell of comprising described one or more different siRNA.Perhaps, with described reagent the effect of the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target is compared, described reagent can weaken the effect of the group of each described one or more cell of comprising described one or more different siRNA.

Preferably, described reagent acts on by fixed arbitrary described different genes gene or its encoded protein in addition of described a large amount of siRNA targets.Preferably, described a large amount of siRNA comprise the fixed different siRNA of at least a described heterogeneic kind of k at least of target, and wherein said k is selected from 2,3,4,5,6 and 10.More preferably, described a large amount of siRNA comprise each the different siRNA of the kind of k at least at least two kinds of different genes in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.Still more preferably, described a large amount of siRNA comprise each the different siRNA of the kind of k at least in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.

Preferably, at least a, at least two kinds or each in described a large amount of different genes all comprises target phasing 2,3,4,5,6 or 10 kind of different siRNA with target gene.In a kind of preferred embodiment, total siRNA concentration of one or more siRNA approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, total siRNA concentration of one or more siRNA is the optimal concentration that is used to make as the target gene silence of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among one or more siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among one or more siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among one or more siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA among one or more siRNA.In a kind of preferred embodiment, select the composition of one or more siRNA, the concentration that comprises number He each siRNA of different siRNA, make one or more siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another embodiment, the optimal concentration the when concentration of each siRNA among one or more siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among one or more siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each the different siRNA among one or more siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and described a large amount of siRNA causes at least 80 or 90% of target gene silence.

In one embodiment, described cell type is the cancer cells type.In another embodiment, described effect is the growth-inhibiting effect.In a kind of specific embodiment, described reagent is the KSP inhibitor.In preferred embodiments, described different gene comprises at least 5 kinds, at least 10 kinds, at least 100 kinds or at least 1000 kinds of different genes.In one embodiment, described different gene is different native gene.

On the other hand, the invention provides the method that is used for identifying with a kind of interactional gene of elementary (primary) target gene of cell of cell type.This method comprises that (a) makes a large amount of group of one or more cells of described cell type contact with a kind of reagent, wherein said reagent is regulated described primary target expression of gene and/or by the proteic activity of described primary target genes encoding, and the group of wherein said cell comprises one or more the different siRNA among a large amount of different siRNA, described one or more different siRNA target phasings gene together, and described a large amount of different siRNA comprise the siRNA of different secondary (secondary) gene in the fixed described cell of difference target; (b) effect to the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; If (c) effect of the cell of the described cell type of the siRNA of arbitrary described different secondary genes is different calmly to not comprising target to the effect of the group of described one or more cells of one or more different siRNA of comprising the fixed described gene of target and described reagent for described reagent, then identified gene is the gene of the described primary target gene interaction in the cell of described and described cell type.In one embodiment, comprise one or more each described groups of cells among described a large amount of siRNA by before described contact procedure, obtaining with described one or more siRNA transfections.

In a kind of specific embodiment, the invention provides the method for the gene of the primary target gene interaction in the cell of identifying with a kind of cell type, described method comprises that (a) is with comprising from a large amount of group of one or more cells of the described cell type of composition transfection of one or more different siRNAs (siRNA) of a large amount of different siRNA each, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target; (b) described a large amount of group of one or more cells of described cell type is contacted with a kind of reagent, wherein said reagent is regulated described primary target expression of gene and/or by the proteic activity of described primary target genes encoding; (c) effect to the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; If (d) effect of the cell of the described cell type of the siRNA of arbitrary described different secondary genes is different calmly to not comprising target to the effect of the group of described one or more cells of one or more siRNA of comprising the fixed described gene of target and described reagent for described reagent, then identified gene is the gene of the described primary target gene interaction in the cell of described and described cell type.

In one embodiment, described reagent comprises the fixed described primary target gene of target and makes its reticent siRNA.In another embodiment, described reagent comprises 2,3,4,5,6 or 10 kind of different siRNA of the fixed described primary target gene of target.In a kind of preferred embodiment, each of described different siRNA is the fixed described primary target gene of target all.In a kind of preferred embodiment, total siRNA concentration of the described different siRNA approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, total siRNA concentration of described different siRNA is the optimal concentration that is used to make the primary target gene silencing.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among the different siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among the different siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among the different siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA among the different siRNA.In a kind of preferred embodiment, select the composition of different siRNA, the concentration that comprises number He each siRNA of different siRNA, make different siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another embodiment, the optimal concentration the when concentration of each siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each different siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and all siRNA cause at least 80 or 90% of target gene silence together.In another embodiment, described reagent comprises the proteic inhibitor by described primary target genes encoding.

With described reagent the effect of the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target is compared, described reagent can strengthen the effect of the group of described one or more cells.Perhaps, with described reagent the effect of the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target is compared, described reagent can weaken the effect of the group of described one or more cells.

Preferably, one or more siRNA at least a, at least two kinds or each in described a large amount of different genes comprise target phasing 2,3,4,5,6 or 10 kind of different siRNA with target gene.In a kind of preferred embodiment, the target phasing is identical with the about concentration with the single siRNA that uses separately of total siRNA concentration of one or more siRNA of target gene, for example 100nM.Preferably, total siRNA concentration of one or more siRNA is the optimal concentration that is used to make as the target gene silence of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among one or more siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among one or more siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among one or more siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA among one or more siRNA.In a kind of preferred embodiment, select the composition of one or more siRNA, the concentration that comprises number He each siRNA of different siRNA, make one or more siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another embodiment, the optimal concentration the when concentration of each siRNA among one or more siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among one or more siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each the different siRNA among one or more siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and described a large amount of siRNA causes at least 80 or 90% of target gene silence.

In one embodiment, the group of each described one or more cell is by obtaining with described one or more different siRNA transfections before described contact procedure.In another embodiment, described primary target is KSP.In preferred embodiments, described different secondary gene comprises at least 5 kinds, at least 10 kinds, at least 100 kinds or at least 1000 kinds of different genes.In one embodiment, described different secondary gene is different native gene.In one embodiment, described cell type is the cancer cells type.

On the other hand, the invention provides and be used for the treatment of the mammiferous method of suffering from cancer, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, wherein said Mammals carries out a kind of treatment, and described treatment comprises the KSP inhibitor to described administration treatment q.s.The present invention also provides and has been used for the treatment of the mammiferous method of suffering from cancer, comprise to described administration i) treatment q.s a kind of reagent, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding with ii) treat the KSP inhibitor of q.s.In one embodiment, described reagent reduces STK6 or TPX2 expression of gene described in the cell of described cancer.In a kind of preferred embodiment, described reagent comprises the siRNA of fixed described STK6 of target or TPX2 gene.In another embodiment, described Mammals is the people, and described siRNA can be selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described TPX2 gene of target.In another embodiment, described Mammals is the people, and described siRNA can be selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQID NO:1239.

In another embodiment, the invention provides and be used for the treatment of the mammiferous method of suffering from cancer, comprise to described administration i) treatment q.s first kind of reagent, described first kind of reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, ii) treat second kind of reagent of q.s, described second kind of reagent is regulated the KSP expression of gene and/or by the proteic activity of described KSP genes encoding.In a kind of preferred embodiment, described first kind of reagent comprises the siRNA of fixed described TPX2 of target or TPX2 gene, and described second kind of reagent comprises the siRNA of the fixed described KSP gene of target.In another kind of preferred embodiment, described Mammals is the people, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described TPX2 gene of target.In another embodiment, described Mammals is the people, and described siRNA can be selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.

In another embodiment, the invention provides and be used to assess the method for cell the Growth Inhibition resistance of KSP inhibitor, described method comprises STK6 or the TPX2 expression of gene level of determining in the described cell, the described expression level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.In a kind of preferred embodiment, by comprising the method for measuring described STK6 or TPX2 expression of gene level with one or more nucleotide probes, determine described STK6 or TPX2 expression of gene level, each in described one or more polynucleotide probes all comprises the nucleotide sequence in described STK6 or the TPX2 gene.Described one or more polynucleotide probes can be the polynucleotide probes on microarray.

In another embodiment, the invention provides and be used to assess the method for cell the Growth Inhibition resistance of KSP inhibitor, described method comprises the proteic abundance level of determining in the described cell by STK6 or TPX2 genes encoding, the described proteic described abundance level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.The present invention also provides and has been used to assess the method for cell to the Growth Inhibition resistance of KSP inhibitor, described method comprises the proteic activity level of determining in the described cell by STK6 or TPX2 genes encoding, the described activity level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.In a kind of preferred embodiment, described cell is people's cell.

In another embodiment, the invention provides and be used to regulate the method for cell the Growth Inhibition resistance of KSP inhibitor, comprise a kind of reagent that makes described cells contacting q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding.The present invention also provides and has been used for regulating the method for Mammals cell to the Growth Inhibition resistance of KSP inhibitor, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding.The present invention also provides the method that is used to regulate the cell growth, comprise and make described cells contacting i) a kind of reagent of q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding and the ii) KSP inhibitor of q.s.Preferably, described reagent reduces STK6 described in the described cell or TPX2 expression of gene.In a kind of preferred embodiment, described reagent comprises the siRNA of the fixed described STK6 gene of target.In another embodiment, described cell is people's cell, and wherein said siRNA is selected from the siRNA shown in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5 and the SEQ ID NO:6.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described TPX2 gene of target.In another embodiment, described cell is people's cell, and described siRNA can be selected from the siRNA shown in SEQ ID NO:1237, SEQID NO:1238 and the SEQ ID NO:1239.

In another embodiment, the invention provides and be used to identify and regulate the compositions and methods of cell the Growth Inhibition resistance of KSP inhibitor, wherein said reagent can be regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, described method comprise described KSP inhibitor when relatively having described reagent to the cell inhibiting effect of expressing described STK6 or TPX2 gene when not having described reagent described KSP inhibitor to expressing the cell inhibiting effect of described STK6 or TPX2 gene, the described inhibiting difference of wherein said KSP inhibitor identifies that described reagent can regulate the Growth Inhibition resistance of described cell to the KSP inhibitor.

The present invention also provides and has been used to identify and can regulates the compositions and methods of cell to the Growth Inhibition resistance of KSP inhibitor, wherein said reagent can be regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, described method comprises: (a) first kind of cell of expressing described STK6 or TPX2 gene contacted existing with described KSP inhibitor, and measure first kind of growth-inhibiting effect; (b) second kind of cell of expressing described STK6 or TPX2 gene contacted with described KSP inhibitor, and measure second kind of growth-inhibiting effect; (c) compare in described step (a) and described first kind and second kind of restraining effect of measuring (b), difference between wherein said first kind and the second kind of restraining effect identifies that described reagent can regulate the Growth Inhibition resistance of cell to the KSP inhibitor.In a kind of preferred embodiment, described reagent is the molecule that reduces described STK6 or TPX2 genetic expression.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described STK6 gene of target.In another kind of preferred embodiment, described cell is people's cell, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described TPX2 gene of target.In another embodiment, described cell is people's cell, and described siRNA can be selected from the siRNA shown in SEQID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.

On the other hand, the invention provides the cell that comprises one or more different siRNAs (siRNA), STK6 or TPX2 gene in the fixed described cell of described siRNA target.Described cell can be people's cell.Described cell also can be the mouse cell.In one embodiment, described cell is people's cell, and among described one or more different siRNA each all is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.In another embodiment, described cell is people's cell, and described siRNA can be selected from the siRNA shown in SEQ IDNO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.In one embodiment, described cell produces by the composition transfection of using described one or more different siRNA, total siRNA concentration of wherein said composition is the optimal concentration that is used to make described STK6 or TPX2 gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, do not increase reticent level substantially.In one embodiment, described optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than more than 20%, more than 10% or 5%.In one embodiment, the concentration of every kind of described different siRNA is approximately identical.In one embodiment, every kind of described different siRNA concentration separately difference each other is less than 50%, less than 20% or less than 10%.In another embodiment, do not have in the described composition a kind of siRNA account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20%.In another embodiment, the concentration of at least a siRNA in the described composition is more than 20% or more than 50% of described total siRNA concentration of described different siRNA.In another embodiment, select the concentration of number He each siRNA of different siRNA in the described composition, make described composition cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

On the other hand, the invention provides and be used for the microarray of diagnosis cell the Growth Inhibition resistance of KSP inhibitor.Described microarray comprises one or more polynucleotide probes, and wherein each described polynucleotide probes comprises the nucleotide sequence in STK6 or the TPX2 gene.

On the other hand, the invention provides and be used for the test kit of diagnosis cell the Growth Inhibition resistance of KSP inhibitor.Described test kit comprises one or more polynucleotide probes that place one or more containers, and wherein each described polynucleotide probes comprises the nucleotide sequence in STK6 or the TPX2 gene.The present invention also provides the test kit that is used to screen a kind of reagent, and described reagent is regulated the Growth Inhibition resistance of cell to the KSP inhibitor.Described test kit comprises the cell that (i) that place one or more containers comprises one or more different siRNAs (siRNA), STK6 or TPX2 gene in the fixed described cell of described siRNA target; (ii) KSP inhibitor.On the other hand, the invention provides and be used for the treatment of the mammiferous test kit of suffering from cancer, this test kit comprises the adjusting STK6 of (i) q.s that places one or more containers or TPX2 expression of gene and/or by the proteic active reagent of described STK6 or TPX2 genes encoding; (ii) KSP inhibitor.

In the present invention, the KSP inhibitor can be (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2 of describing among the PCT application PCT/US03/18482 that submitted on June 12nd, 2003,5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine.

The present invention also provides the method that is used for identifying with a kind of gene of primary target gene interaction of cell of cell type.This method comprises that (a) makes a kind of reagent of one or more cells contacting of described cell type, wherein said reagent is regulated the secondary target expression of gene and/or by the proteic activity of described secondary target genes encoding, and first siRNA (siRNA) of the fixed described primary target gene of wherein said one or more cell expressing targets; (b) more described reagent is to the effect to the cell of the described cell type of not expressing a described siRNA of the effect of described one or more cells of described clone and described reagent; If (c) described reagent is different to the effect of the cell of the described cell type of not expressing a described siRNA to the effect of described one or more cells of expressing a described siRNA and described reagent, then identify described secondary target gene for the cell of described cell type in the gene of primary target gene interaction.

In a kind of particular, described method comprises that (a) produces the expression target clone of the cell of the described cell type of first siRNA (siRNA) of described primary target gene calmly; (b) make a kind of reagent of one or more cells contacting of described clone, wherein said reagent is regulated the secondary target expression of gene and/or by the proteic activity of described secondary target genes encoding; (c) more described reagent is to the effect to the cell of the described cell type of not expressing a described siRNA of the effect of described one or more cells of described clone and described reagent; If (d) described reagent is different to the effect of the cell of the described cell type of not expressing a described siRNA to the effect of described one or more cells of expressing a described siRNA and described reagent, then identify described secondary target gene for the cell of described cell type in the gene of primary target gene interaction.

In some embodiments, with described reagent the effect of the cell of the described cell type of not expressing a described siRNA is compared, described reagent strengthens the effect of described one or more cells of expressing a described siRNA.In some embodiments, with described reagent the effect of the cell of the described cell type of not expressing a described siRNA is compared, described reagent weakens the effect of described one or more cells of expressing a described siRNA.In one embodiment, described reagent is the inhibitor of described secondary target gene.The effect of described reagent can be the change of the cell of described cell type to the susceptibility of medicine or ionizing rays, described medicine such as DNA disrupting agent are as the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum and antimetabolite.

In another embodiment, described reagent comprises the fixed described secondary target gene of one or more targets and makes the 2nd siRNA of its silence.Preferably, described one or more comprise the different siRNA of k kind at least, as at least 2,3,4,5,6 or 10 kind of different siRNA.In a kind of preferred embodiment, total siRNA concentration of one or more the 2nd siRNA approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, total siRNA concentration of one or more the 2nd siRNA is the optimal concentration that is used to make as the secondary target gene silencing of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among one or more the 2nd siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among one or more the 2nd siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among one or more the 2nd siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more the 2nd siRNA among one or more siRNA.In a kind of preferred embodiment, select the composition of one or more siRNA, the concentration that comprises number He each the 2nd siRNA of different siRNA, make one or more the 2nd siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another embodiment, the optimal concentration the when concentration of each siRNA among one or more the 2nd siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among one or more siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each the different siRNA among one or more the 2nd siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the secondary target gene silencing that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and described a large amount of siRNA causes at least 80 or 90% of secondary target gene silencing.

In one embodiment, described cell is the cancer cells type.In another embodiment, described primary target gene is p53.

In a kind of preferred embodiment, in a large amount of different secondary target genes each, the step of repetition methods (b)-(d).A large amount of secondary target genes can comprise at least 5,10,100,1000 with 5000 kinds of different genes.

The present invention also provides and has been used for the treatment of the mammiferous method of suffering from cancer.This method comprises a kind of reagent to described administration treatment q.s, described reagent is regulated a kind of expression of gene and/or by the proteic activity of described genes encoding, wherein said Mammals carries out a kind of treatment, and described treatment comprises the composition that comprises one or more DNA disrupting agents to described administration treatment q.s.In one embodiment, the invention provides and be used for the treatment of the mammiferous method of suffering from cancer, comprise to described administration i) treatment q.s a kind of reagent, described reagent is regulated a kind of expression of gene and/or by the proteic activity of described genes encoding with ii) treat the composition that comprises one or more DNA disrupting agents of q.s.

Preferably, described reagent makes the expression decreased of described gene in the cell of described cancer.In a kind of preferred embodiment, described reagent comprises the siRNA of the fixed described gene of target.In specific embodiments, described gene is EPHB3, WEE1, ELK1, STK6, CHEK1 or BRCA2.Described reagent also can be to strengthen the reagent that described gene is expressed in the cell of described cancer.Described one or more DNA disrupting agents can comprise the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.

The present invention also provides and has been used to assess the method for cell to the Growth Inhibition susceptibility of DNA disrupting agent.Described method comprises a kind of gene transcription level of determining in the described cell, wherein is lower than the described transcriptional level of predetermined threshold levels, shows that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.Described DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.In a kind of preferred embodiment, described gene is EPHB3, WEE1, ELK1, STK6, CHEK1 or BRCA2.In a kind of preferred embodiment, by comprising the method for measuring described gene transcription level with one or more nucleotide probes, determine described gene transcription level, each in described one or more polynucleotide probes all comprises the nucleotide sequence in the described gene.In one embodiment, described one or more polynucleotide probes are the polynucleotide probes on microarray.

In another embodiment, the invention provides and be used to assess cell, as the method for people's cell to the Growth Inhibition susceptibility of DNA disrupting agent.Described method comprises to be determined in the described cell wherein to be lower than the described proteic described abundance level of predetermined threshold levels by a kind of proteic abundance level of genes encoding, shows that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.The present invention also provides and has been used to assess cell, as the method for people's cell to the Growth Inhibition susceptibility of DNA disrupting agent, described method comprises the proteic activity level of determining in the described cell by described genes encoding, the described activity level that wherein is higher than predetermined threshold levels shows that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.The DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.In a kind of preferred embodiment, described gene is EPHB3, WEE1, ELK1, STK6, CHEK1 or BRCA2.

The present invention also provides and has been used to regulate the method for cell to the susceptibility of DNA disrupting agent.This method comprises makes described cell contact with a kind of reagent of q.s, and described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding.The present invention also provides the method that is used to regulate the cell growth, comprise and make described cells contacting i) a kind of reagent of q.s, described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding; The ii) DNA disrupting agent of q.s.The DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.

In one embodiment, described reagent reduces expression of gene described in the described cell.In a kind of preferred embodiment, described reagent comprises the siRNA of the fixed described gene of target.In another kind of preferred embodiment, described reagent comprises 2,3,4,5,6 or 10 kind of different siRNA of the fixed described gene of target.In a kind of preferred embodiment, total siRNA concentration of the different siRNA of the fixed described gene of the target approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, the total concn of the different siRNA of the fixed described gene of target is the optimal concentration that is used to make described gene silencing.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among the different siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among the different siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among the different siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA among the different siRNA.In a kind of preferred embodiment, select the composition of different siRNA, the concentration that comprises number He each siRNA of different siRNA, make different siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.Optimal concentration when in other embodiments, the concentration of each siRNA among the different siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among the different siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each different siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the gene silencing that the concentration of every kind of siRNA causes causes when being lower than independent the use, and a large amount of siRNA causes at least 80 or 90% of target gene silence.

The present invention also provides and has been used to identify and can regulates the compositions and methods of cell to the Growth Inhibition susceptibility of DNA disrupting agent, wherein said reagent can be regulated and be selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, the expression of gene of BARD1 and RAD51 and/or by the proteic activity of described genes encoding, this method comprise described DNA disrupting agent when relatively having described reagent to the cell inhibiting effect of expressing said gene when not having described reagent described DNA disrupting agent to the cell inhibiting effect of expressing said gene, the described inhibiting difference of wherein said DNA disrupting agent identifies that described reagent can regulate the Growth Inhibition susceptibility of described cell to the DNA disrupting agent.In one embodiment, the invention provides and be used to identify and regulate the compositions and methods of cell the Growth Inhibition susceptibility of DNA disrupting agent, wherein said reagent can be regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding, this method comprises that (a) exists the cell that makes first kind of expressing said gene under the condition of described reagent to contact with described DNA disrupting agent, and measures first kind of growth-inhibiting effect; (b) cell of second kind of expressing said gene is contacted with described DNA disrupting agent, and measure second kind of growth-inhibiting effect; (c) compare in described step (a) and described first kind and second kind of restraining effect of measuring (b), difference between wherein said first kind and the second kind of restraining effect identifies that described reagent can regulate the Growth Inhibition susceptibility of described cell to the DNA disrupting agent.

Preferably, described cell expressing target is decided the siRNA of primary target gene.In one embodiment, described primary target gene is p53.

In a kind of preferred embodiment, described reagent is the molecule that reduces described genetic expression.In a kind of preferred embodiment, described reagent comprises the siRNA of the fixed described gene of target.In another kind of preferred embodiment, described reagent comprises 2,3,4,5,6 or 10 kind of different siRNA of the fixed described gene of target.In a kind of preferred embodiment, total siRNA concentration of the different siRNA of the fixed described gene of the target approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, the total concn of the different siRNA of the fixed described gene of target is the optimal concentration that is used to make described gene silencing.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among the different siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among the different siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among the different siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA among the different siRNA.In a kind of preferred embodiment, select the composition of different siRNA, the concentration that comprises number He each siRNA of different siRNA, make different siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In other scheme, the optimal concentration the when concentration of each siRNA among the different siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among the different siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each different siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the gene silencing that the concentration of every kind of siRNA causes causes when being lower than independent the use, and a large amount of siRNA causes at least 80 or 90% of target gene silence.

In described method, described DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.

The present invention also provides the cell that comprises one or more different siRNAs (siRNA), the target gene that is selected from EPHB3, WEE1, ELK1, BRCA1, BRCA2, BARD1 and RAD51 in the fixed described cell of described siRNA target.In one embodiment, described one or more siRNA comprise 2,3,4,5,6 or 10 kind of different siRNA.In a kind of preferred embodiment, total siRNA concentration of described one or more siRNA approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, the total concn of described one or more siRNA is the optimal concentration that is used to make as the target gene silence of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among described one or more siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among described one or more siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among described one or more siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of described one or more siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of described one or more siRNA among described one or more siRNA.In a kind of preferred embodiment, select the composition of described one or more siRNA, the concentration that comprises number He each siRNA of different siRNA, make described one or more siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In other scheme, the optimal concentration the when concentration of each siRNA among described one or more siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among described one or more siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each the different siRNA among described one or more siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and a large amount of siRNA causes at least 80 or 90% of target gene silence.

The present invention also provides and has been used for the microarray of diagnosis cell to the Growth Inhibition susceptibility of DNA disrupting agent.Described microarray comprises one or more polynucleotide probes, and wherein each described polynucleotide probes comprises the nucleotide sequence in one or more genes that are selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

The present invention also provides and has been used for the test kit of diagnosis cell to the Growth Inhibition susceptibility of DNA disrupting agent.Described test kit comprises one or more polynucleotide probes that place one or more containers, and wherein each described polynucleotide probes comprises the nucleotide sequence of the gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

The present invention also provides and has been used to screen the test kit of adjusting cell to the reagent of the Growth Inhibition susceptibility of DNA disrupting agent.Described test kit comprises the cell that (i) that place one or more containers comprises one or more different siRNAs (siRNA), the gene that is selected from EPHB3, WEE1, ELK1, BRCA1, BRCA2, BARD1 and RAD51 in the fixed described cell of described siRNA target; (ii) described DNA disrupting agent.

The present invention also provides and has been used for the treatment of the mammiferous test kit of suffering from cancer, described test kit comprises a kind of reagent of (i) q.s that places one or more containers, and described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding; (ii) described DNA disrupting agent.

In test kit of the present invention, described DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.

The present invention also provides and has been used to assess the reactive method of a kind of cell of cell type to pharmacological agent, comprise that (a) makes the described medicine of one or more cells contacting of described cell type, wherein said one or more cell expressing targets are decided first siRNA (siRNA) of primary target gene, and wherein said one or more cells are accepted a kind of processing of composition, and described composition is regulated one or more secondary target expression of gene and/or respectively by the proteic activity of described one or more secondary target genes encodings; (b) make the described medicine of one or more cells contacting of described cell type, wherein said one or more cells are not expressed the siRNA (siRNA) of the fixed described primary target gene of target, and wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active described reagent of described secondary target genes encoding; (c) the described medicine of relatively measuring in step (a) is to the effect to described one or more cells of the described medicine of the effect of described one or more cells and measurement in step (b), thereby assesses the reactivity of described cell to described pharmacological agent.In one embodiment, described method further comprises the step (d) to each repeating step (a)-(b) in the different in a large number secondary target genes.

In a kind of particular, the invention provides and be used to assess the reactive method of a kind of cell of cell type to pharmacological agent, this method comprises that (a) preparation expresses the clone of cell of described subclass type that target is decided first siRNA (siRNA) of primary target gene; (b) make the described clone's who expresses a described siRNA the described medicine of one or more cells contacting, wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active reagent of described secondary target genes encoding; (c) make the described medicine of one or more cells contacting of the described cell type of the siRNA (siRNA) of not expressing the fixed described primary target gene of target, wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active described reagent of described secondary target genes encoding; (d) the described medicine of relatively measuring in step (b) is to the effect to described one or more cells of the described medicine of the effect of described one or more cells and measurement in step (c), thereby assesses the reactivity of described cell to described pharmacological agent.In one embodiment, described method further comprises the step to each repeating step (b)-(d) in the different in a large number secondary target genes.

In one embodiment, with described medicine the effect of the cell of the described cell type of not expressing a described siRNA is compared, described medicine strengthens the effect of one or more cells of expressing a described siRNA.In another embodiment, with described medicine the effect of the cell of the cell type of not expressing a described siRNA to be compared, described medicine weakens the effect of one or more cells of expressing a described siRNA.

In one embodiment, described composition comprises one or more inhibitor of described one or more secondary target genes.In a kind of preferred embodiment, described composition comprises fixed described one or more second target genes of target and makes one or more the 2nd siRNA of its silence.

In one embodiment, described one or more the 2nd siRNA comprise the different siRNA of k kind at least, and wherein said k is selected from 2,3,4,5,6 and 10.In one embodiment, the total siRNA concentration of the different siRNA of the described kind of k at least in the described reagent is the optimal concentration that makes described secondary target gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, described optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than more than 20%, more than 10% or 5%.In another embodiment, the concentration of each of the different siRNA of the described kind of k at least is approximately identical.In another embodiment, the different siRNA concentration separately of k kind difference each other is less than 50%, less than 20% or less than 10% at least.In another embodiment, do not have in the described reagent a kind of siRNA account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20%.In another embodiment, the concentration of at least a siRNA in the described reagent is more than 20% or more than 50% of described total siRNA concentration of the different siRNA of the described kind of k at least.In another embodiment, select the concentration of number He each siRNA of different siRNA in the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

In some embodiments, described cell is the cancer cells type, and described primary target gene is p53.In preferred embodiments, described a large amount of secondary target gene comprise at least be selected from down the group heterogeneic number: 5,10,100,1000 with 5000 kinds of different genes.

In one embodiment, described medicine is the DNA disrupting agent, as is selected from the DNA disrupting agent of topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.In a kind of specific embodiment, described DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.

4. accompanying drawing summary

Fig. 1 represents the mRNA silence of STK6 and the association between the growth-inhibiting phenotype.With six kinds of STK6 independent siRNA transfection HeLa cells.After transfection 24 hours, gather in the crops one group of cell and be used for RNA and separate, and determine the STK6mRNA level by adopting the TaqMan that measures (Assay onDemand) (Applied Biosciences) as required to analyze.Further another group cell (72 hours altogether) of incubation, and employing Alamar Blue is determined at assessment cell growth in three parts of holes.The rna level (X-axis) of each part and the value of cell growth (Y-axis) are carried out normalization method, so that the contrast of simulation transfection.Analyze for TaqMan, each data point representative is with the single rna sample (and normalizing to GUS) of three parts of mensuration; Variation between the repeated experiments usually＜10%.For the determined value of growth measurement, each data point is represented the mean value of three replication values, the common departure of described measured value＜20%.Solid line is represented 1: 1 relation of ideal between silence and the phenotype.

Fig. 2 represents that collaborative the causing death between STK6 and the KSP interacts.With luciferase (negative control) that increases concentration and the siRNA transfection HeLa cell of STK6 (last figure) or PTEN (figure below), and in three days Alamar Blue mensuration, test with respect to the growth that contrasts (luciferase processing).In the place of pointing out, cell is also used 25nM KSPi, (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-processing of 2-methyl propylamine; The EC50 of the HeLa cell of Ce Dinging is about 80nM under these conditions.Shown is the mean value ± SD (error post) of three replication values.

Fig. 3 has proved that the stably express of TP53shRNA makes the target gene silence effectively.Decide shRNA plasmid (pRS-p53) the transfection HCT116 cell of TP53 with target.What illustrate is wild-type (WT) cell and with the TP53mRNA level in two independent clonings (A5 and A11) of the cell of pRS-p53 stable transfection.Silence＞95% (intermediary post) of TP53mRNA level in clone A5 and A11.In the instantaneous importing HCT116 of pRS-p53 cell, after transfection, reached about 80% silence (post on the right) in 24 hours.

After Fig. 4 is illustrated in the siRNA excess revolutions and dyes, express the silence of keeping mRNA by stable shRNA.(A) pRS-p53 does not influence the silence of the CHEK1 that siRNA causes, and vice versa.The set transient transfection of three kinds of siRNA of target being decided CHEK1 is to the HCT116 cell of WT and pRS-p53 stable transfection (clone A11).Analyze (being respectively left figure and right figure) by Taqman and measure CHEK1 and TP53mRNA level.(B) KNSL1siRNA that dyes of the excess revolutions silence that causes of competitive inhibition pRS-STK6 not.STK6 and KNSL1siRNA transient cotransfection in WT SW480 cell, and are dyed the KNSL1siRNA excess revolutions in the SW480 cell of pRS-STK6 stable transfection.By Taqman analysis to measure STK6mRNA level.For one group of post on the left side, use separately or the single KNSL1 siRNA (every kind 10nM) different with three kinds in a kind of STK6 siRNA (10nM) that uses together.KNSL1 siRNA suppresses the silence that STK6 siRNA causes changeably.For two groups of posts on the right, with 10 or 100nM with the competitor of KNSL1 siRNA as the STK6shRNA of stably express.

Fig. 5 has proved the siRNA library screening that does not exist under the DNA destructive condition, shows that having and do not have target decides good correlation between the cell of shRNA of p53.With pRS (the carrier is only arranged) cell (x axle) that the set excess revolutions of three kinds of siRNA is dyed, each of described three kinds of siRNA is one of fixed 800 kinds of genes of target all, and the relevant phenotype of test vector generation for testing IC; Measure pRS-p53 cell (y axle) in an identical manner.Substantial connection between two groups of data shows that the performance of siRNA set is not subjected to the influence of the existence of shRNA probably, shows that shRNA does not compete with siRNA.

The CHEK1 silence in the pRS-p53 cell of representing Fig. 6 has reduced the G2 outpost of the tax office and has stagnated (checkpoint arrest).Only use carrier (pRS) stable transfection A549 cell or dye the pRS-p53 cell with the set excess revolutions of three kinds of siRNA that contrast (luc, luciferase) siRNA or CHEK1.After transfection, added Zorubicin (200ng/ml) in 24 hours, and after adding Zorubicin 48 hours analysis of cells cyclic spectrums.Compare with the pRS cell, the TP53mRNA level in the pRS-p53 cell has reduced about 90%.

Fig. 7 has illustrated the evaluation to the gene of cis-platinum sensitivity.SiRNA with the about 800 kinds of people's genes of representative gathers the HeLa cell that (3siRNAs/ gene, total siRNA concentration is 100nM) transfection is grown on 384 orifice plates.After the transfection four hours, handle cell with independent substratum (or adding excipient) (medicine) or the substratum that adds the cis-platinum (Cis ,+medicine) of EC10 concentration.72 hours measurement cells are grown after being determined at transfection with Alamar Blue then, and are expressed as the growth % that measures in the hole with luciferase siRNA transfection.Each point is represented the mean number of 2-4 replication value.

Fig. 8 represents the comparison to the gene of different pharmacological agent sensitivities.By the siRNA transfection HeLa cell of use shown in Figure 1, and with independent substratum (or adding excipient) or add the substratum processing cell of Cis, Zorubicin (Dox) or the camptothecine (Campto) of EC10 concentration.The measurement cell is grown, and is expressed as the ratio of growth-medicine/growth+medicine.Dotted line is represented twice sensitization.Pointed out selected gene.

Fig. 9 A-9C represents that the silence of WEE1 makes the HeLa cell destroy responsive to Dox, Campto and Cis inductive DNA.Fig. 9 D-9I shows that the silence of WEE1 makes the p53-A549 cell destroy sensitivity to Dox, Campto and Cis inductive DNA, but it is responsive that the p53+A549 cell is destroyed described DNA.

Figure 10 A-10C shows that the silence of EPHB3 makes HeLa cell and p53-A549C7 destroy sensitivity to Dox, Campto and Cis inductive DNA, makes the p53+A549pRS cell lower to the degree of Dox, Campto and Cis inductive DNA destruction sensitivity.

Figure 11 A-11C shows that the STK6 silence makes HeLa cell and p53-A549C7 destroy sensitivity to Dox, Campto and Cis inductive DNA, makes the p53+A549pRS cell lower to the degree of Dox, Campto and Cis inductive DNA destruction sensitivity.

Figure 12 A-12C shows that the silence of BRCA1 makes HeLa cell and p53-A549C7 cell destroy responsive to Dox, Campto and Cis inductive DNA.It is responsive that the silence of BRCA also makes the p53+A549pRS cell destroy Cis inductive DNA with lower degree, but do not make the p53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.

Figure 13 A-13B shows that the silence of BRCA2 makes HeLa cell and p53-A549C7 cell destroy responsive to Dox, Campto and Cis inductive DNA.It is responsive that Figure 13 C shows that the silence of BRCA makes the p53+A549pRS cell destroy Cis inductive DNA with lower degree, but do not make the p53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.

Figure 14 A-14B shows that the silence of CHUK makes the HeLa cell destroy responsive to Dox, Campto and Cis inductive DNA.Figure 14 C shows that the silence of CHUK makes the p53-A549pRS cell destroy responsive to Campto and Cis inductive DNA.Figure 14 D shows that the silence of CHUK does not make the 53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.

Figure 15 A-C shows the result of the reticent pair cell of CHEK1 to the influence of DNA destructive susceptibility.It is responsive that 15A:CHEK1 silence/inhibition makes the HeLa cell destroy DNA.It is responsive that 15B:CHEK1 silence/inhibition makes the p53-A549 cell destroy DNA.The 15C:CHEK1 silence does not make the HREP cell to the Zorubicin sensitivity.

Figure 16 represents that knock out (knockdown) of the PLK gene of siRNA mediation caused cell cycle arrest and apoptosis.

Figure 17 represents to screen the result to the gene of KSPi sensitivity.

Figure 18 represents to screen the result to the gene of taxol sensitivity.

Figure 19: the BRCA mixture has strengthened the cis-platinum activity., use cis-platinum (Y-axis) then or handle with the siRNA of about 2000 kinds of genes set (3siRNAs/ gene) transfection HeLa cell with 384 well format without cis-platinum (X-axis).Detect two kinds of different cis-platinum concentration, i.e. 100ng/ml (about EC10, left figure) or 400ng/ml (about EC50, right figure).After transfection 72 hours, measure the cell growth with Alamar Blue assay method.Diagonal lines is represented the consistence between twice processing (black line), or 2 times and 3 times of sensitization (being respectively fuchsin and red line) by cisplatin treated.

It is responsive that the silence of Figure 20: BRCA1 preferentially makes the TP53-cell that DNA is destroyed.With empty carrier (pRS, left figure) stable transfection A549 cell, or dye target with the siRNA excess revolutions of luciferase, BRCA1 or BRCA2 and decide the shRNA of TP53 (pRS-TP53, right figure), use DNA disrupting agent cisplatin treated then.After transfection 72 hours, measure the cell growth with Alamar Blue.

It is responsive that the reticent selectivity of Figure 21: BRCA1 makes the TP53-cell destroy DNA.With TP53 feminine gender (left column) or the positive (right row) the A549 cell that the siRNA transfection of luciferase (top line) or BRCA1 (end row) is mated, handle with DNA disrupting agent bleomycin then.After the transfection 72 hours, fixed cell, with the dyeing of iodate third ingot, and by the flow cytometry cell cycle distribution.Relative fluorescence with cell of 2N or 4N dna content is represented with arrow.The grid that marks with redness shows the number of inferior G1 (death) cell.

The result that Figure 22 represents proves that RAD51/ Zorubicin synergy is bigger in the TP53-cell.

5. detailed Description Of The Invention

The invention provides and adopt RNA to disturb identified gene or its product and a kind of reagent, for example interaction between the medicine is as causing death/the collaborative interactional method and composition that causes death.The term of Shi Yonging " gene product " comprises from the mRNA of genetic transcription with by the albumen of genes encoding herein.The present invention also provides and has utilized STK6 kinases (being also referred to as Aurora A kinases) and KSP (kinesin sample dynein is also referred to as KNSL1 or EG5) inhibitor (KSPi ' s) treatment method for cancer and composition.In this disclosure, use KSPi (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2 usually, 5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine

(the PCT application PCT/US03/18482 referring to submitting on June 12nd, 2003 is incorporated herein by reference in full at this).Other KSPi ' s also can be used for the present invention.Can predict and use the method for described other KSPi ' s to be also included within the scope of the present invention.The present invention also provides the interaction that utilizes DNA to destroy between response gene and the DNA disrupting agent to treat method for cancer and composition.

5.1. disturb the interactional method of screening with RNA

The invention provides in the cell of identifying a kind of cell type and a kind of reagent, for example drug interaction, the method for for example regulating one or more genes of its effect.The interaction of the gene of Shi Yonging and a kind of reagent or another kind of gene herein comprises the interaction of gene and/or its product and this reagent or another kind of gene/gene product.For example, genes identified can be given the resistance of medicine or susceptibility,, reduces or strengthen the effect of medicine that is.Can adopt a large amount of siRNAs (each is one of a large amount of surely different genes of target all) to knock out a large amount of different genes (knocking out cell) in the cell of described cell type, and which kind of or which the generegulation cell in a large amount of different genes of determining to knock out is to the reaction of described reagent, thereby identifies described one or more genes.In one embodiment, a large amount of different cells (knocking out the library) that knock out of preparation knock out in the library each and knock out cell and all comprise the different genes that is for example knocked out by siRNA.In another embodiment, a large amount of different cells (knocking out the library) that knock out of preparation knock out in the library each and knock out cell and all comprise two or more different genes that for example knocked out by fixed heterogeneic shRNA of target and siRNA.In one embodiment, knock out the library and comprise a large amount of cells, wherein each is all expressed the siRNA that target is decided primary gene, and dyes with one or more siRNA excess revolutions of the fixed secondary gene of target.It will be apparent to one skilled in the art that also and can pass through other method, for example knock out cell by using target to decide the antisense molecule of gene or its product, ribozyme, antibody or the preparation of little organic or inorganic molecule.Can predict, any one in described other method and utilize the method for siRNA to be used singly or in combination is to prepare the library that knocks out of the present invention.Can use the method for any siRNA of being used for silence, keep the method for the reticent level that allows the adjusting target gene.Hereinafter 5.2 joints have been described operable several different methods.

In one embodiment, the siRNA that employing comprises a kind of a large amount of cells of cell type knocks out the library and implements method of the present invention, described every kind of cell all comprises one of a large amount of siRNA, among described a large amount of siRNA each all target is decided in the cell (promptly knocking out cell) one of a large amount of different genes and is made its silence (that is, knocking out).Any known method with the siRNA transfered cell can be used for this purpose.Preferably, prepare each cell in a large amount of cells, and keep independently, so that can independently study them.Use each cell in a large amount of cells of a kind of agent treated then, determine the effect of reagent this cell.Then with reagent to comprising by the effect of the cell of the gene of siRNA silence and this reagent to not comprising the cell of siRNA, i.e. the Normocellular effect of this cell type is compared.Identified and reacted on reagent and show the cell that knocks out of change.The described gene that knocks out in the cell by the siRNA silence that comprises is a gene of regulating the effect of described reagent.Preferably, a large amount of siRNA comprise target and decide at least 5 in the cell, 10,100 or 1000 kind of different gene and make its reticent siRNA.In a kind of preferred embodiment, described a large amount of siRNA targets are decided native gene and are made its silence.

In a kind of preferred embodiment, knock out the library and comprise a large amount of different cells that knock out, described cell has the identical gene that knocks out, and for example, it is homogenic and make its reticent siRNA that every kind of cell has different target phasings.Have the identical a large amount of different cells that knock out that knock out gene and can comprise at least 2,3,4,5,6 or 10 kind of different cell that knocks out, wherein each all comprises the siRNA that target knocks out the different zones of gene surely.In another kind of preferred embodiment, knock out the library and comprise a large amount of different cells that knock out, as, at least 2,3,4,5,6 or 10 kind, its at knock out in the library a large amount of heterogeneic each.In another kind of preferred embodiment, knock out the library and comprise a large amount of different cells that knock out, as, at least 2,3,4,5,6 or 10 kind, its at knock out in the library all heterogeneic each.

In another kind of preferred embodiment, knock out the library and comprise a large amount of different different cells that knock out that knock out gene that have, every kind different knocks out cell to have a target phasing homogenic and make two or more different siRNA of its silence.In preferred embodiments, each is different knocks out at least 2,3,4,5,6 or 10 kind of different siRNA that cell can comprise the isogenic different zones of target phasing.

In a kind of preferred embodiment, knock out the reaction assessment gene of cell and the interaction of reagent according to having a large amount of differences that knock out gene, for example, it is homogenic and make the different siRNA of its silence that every kind of cell has the target phasing.Utilize the reaction of a large amount of different siRNA, make it possible to determine that different siRNA are to the effect of target and non-target (referring to, the international application No.PCT/US2004/015439 of the Jackson that submitted on May 17th, 2004 etc. for example).

Compare with the normal cell of described cell type, reagent knocks out in the cell and can weaken the acting on of cell of described cell type, that is, gene knock out the effect that has weakened reagent.The described gene that is knocked out in described cell is considered to give the susceptibility to reagent.Therefore, in one embodiment, method of the present invention is used to identify one or more genes of giving the susceptibility of reagent.

Compare with the normal cell of described cell type, reagent knocks out in the cell and can strengthen the acting on of cell of described cell type.The described gene that is knocked out in described cell is considered to give the resistance to reagent.Therefore, in another embodiment, method of the present invention is used to identify one or more genes of giving the resistance of reagent.Enhancing to the reagent effect can be synergetic or collaborative.In one embodiment, the invention provides and can regulate and/or strengthen anticarcinogen in the cancer cells, as, one or more genes of the Growth Inhibition of KSP inhibitor in the cancer cells.

Method of the present invention can be used to assess a large amount of different reagent.For example, can be by the susceptibility of method assessment of the present invention to a large amount of different DNA disrupting agents of description in the 5.4.2 joint hereinafter.In a kind of preferred embodiment, assessment knocks out every kind of susceptibility that knocks out cell to each reagent in a large amount of different reagent in the library, to produce the reaction matrix of two dimension, wherein comprises every kind of reagent to every kind of observed value that knocks out the effect of cell.The intercept of gene axle under the specific gene index has produced the specific response curve that knocks out cell (wherein specific gene is knocked out) to different pharmaceutical.The intercept of the medicine axle under the certain drug condition has produced the gene response curve to medicine,, comprises that medicine knocks out the curve of observed value of the effect of cell to knocking out difference in the library that is.Table II A-IIC is the example to the gene response curve of cis-platinum (Table II A), camptothecine (Table II B) and Zorubicin (Table II C).

Method of the present invention can be used to identify the interaction between different genes, and this is by use regulating, and for example prevents or reinforcing gene expression and/or realized by the proteic active reagent of genes encoding.The example of described reagent includes, but not limited to target and decides the siRNA of gene or its product, antisense molecule, ribozyme, antibody and little organic or inorganic molecule.The fixed gene of described reagent place target is known as primary target.Described reagent can be used in combination with knocking out the library, regulates the gene of cell to the reaction of reagent to identify.Described primary target can be different from any one that knocks out a large amount of genes (secondary gene) of showing in the library.Therefore the gene that is accredited as the effect of regulating reagent is and the interactional gene of primary target.

In a kind of preferred embodiment, the invention provides the interactional method of using between the dual siRNA method evaluation different genes.In a kind of preferred embodiment, dual RNAi screening is to pass the excess revolutions of deciding the siRNA of secondary target gene with target and dye and realize by sending in the liptinite that uses the shRNA that destroys the primary target gene.This method provides coupling (isogenic) clone to (shRNA adds deduct), and does not cause the competition between shRNA and the siRNA.In the method, the recombinant vectors from instantaneous importing or stable integration to genome express short hairpin RNA (shRNA) (referring to, Paddison et al. for example, 2002, GenesDev 16:948-958; Sui et al., 2002, Proc Natl Acad Sci USA 99:5515-5520; Yu et al., 2002, Proc Natl Acad Sci USA 99:6047-6052; Miyagishi et al., 2002, Nat Biotechnol 20:497-500; Paul et al., 2002, Nat Biotechnol 20:505-508; Kwak et al., 2003, J Phannacol Sci 93:214-217; Brummelkamp et al., 2002, Science 296:550-553; Bodenetal., 2003, Nucleic Acids Res 31:5033-5038; Kawasaki et al., 2003, Nucleic Acids Res 31:700-707).Can express the siRNA that (through shRNA) destroys primary gene by any suitable carrier of coding shRNA.This carrier also can be encoded and be can be used for selecting the marker of cloning, and among the described clone carrier or its enough is partially integrated in the host genome, so that express shRNA.Any standard method well known in the art can be used for carrier sent and is delivered to cell.In one embodiment, by preparing the cell of expressing shRNA with the suitable cell of carrier-containing plasmid transfection.Can select cell by suitable marker then.Selected clone then detects being used to and knocks out.In a kind of preferred embodiment, the expression of shRNA is subjected to the control of inducible promoter, the feasible silence that can open its target gene when needed.The inducible expression of siRNA is specially adapted to the fixed necessary gene of target.

In one embodiment, the control of the promotor that the expression of shRNA is subjected to being regulated, described promotor makes it possible to regulate the reticent level of target gene.This makes it possible to screen specific cells, and the target gene in the described cell is partly knocked out.As used herein, " promotor of being regulated " is meant the promotor that can be activated when suitable inductor exists." inductor " is meant and anyly can be used for the promotor of being regulated by activation and the molecule of activated transcription.Inductor can be, but be not limited to peptide or polypeptide, hormone or organic molecule.Also can use the analogue of inductor, that is, activate the molecule of the promotor of being regulated as inductor.The activity level of the promotor that different analogue inductive are regulated can be different, thereby the adjusting of the activity level of feasible promotor of being regulated is more flexible.The promotor of being regulated in the carrier can be any Mammals transcriptional regulatory well known in the art system (referring to, for example, Gossen et al, 1995, Science 268:1766-1769; Lucas et al, 1992, Annu.Rev.Biochem.61:1131; Li et al., 1996, Cell 85:319-329; Saez et al., 2000, Proc.Natl.Acad.Sci.USA 97:14512-14517; With Pollock et al., 2000, Proc.Natl.Acad.Sci.USA 97:13221-13226).In preferred embodiments, the promotor of being regulated with dosage and/or analogue dependency mode.In one embodiment, by comprising the method for the concentration that the concentration adjustment of inductor can be reacted to the promotor of being regulated, the activity level of the promotor of being regulated is adjusted to the level that needs.Can be according to the reticent level of required target gene, determine the desired level of the promoter activity of being regulated that obtains by the inductor of using specific concentrations.

In one embodiment, use the gene expression system that tsiklomitsin regulates (referring to, Gossen et al for example, 1995, Science 268:1766-1769; U.S. Patent No. 6,004,941).The system that tet regulates uses the composition of procaryotic tet repressor/operon/inductor system to regulate genetic expression in the eukaryotic cell.Like this, the invention provides the expression of using the tet regulation system to regulate the shRNA that is connected with one or more tet operon sequence.This method comprises the carrier transfered cell with the fusion rotein of coding activated transcription.Fusion rotein is included in tsiklomitsin or tetracycline analogue and exists down and tet operon sequence bonded first polypeptide, and described tsiklomitsin or tetracycline analogue are connected with the second polypeptide operability of transcribing in the activating cells.By regulating the concentration of tsiklomitsin or tetracycline analogue, regulate the expression of the shRNA of tet operon connection.

In other embodiments, can use the gene expression system that moulting hormone regulates (referring to, for example, Saez et al., 2000, Proc.Natl.Acad.Sci.USA 97:14512-14517) or the gene expression system regulated of MMTV glucocorticoid responsive element (referring to, for example, Lucas et al, 1992, the Annu.Rev.Biochem.61:1131) expression of adjusting shRNA.

In one embodiment, use coding tetracycline resistance markers and driving pRETRO-SUPER (pRS) carrier from H1 (RNA Pol III) promoter expression shRNA.Can prepare the pRS-shRNA plasmid by standard method well known in the art.In one embodiment, pRS-shRNA is from library plasmid set deconvolution, and this is by gathering transform bacteria with this, and seeks the clone who only contains interested plasmid.Preferably, 19 aggressiveness siRNA sequences are used with the suitable forward and the reverse primer that are used for sequence-specific PCR.Identify plasmid by sequence-specific PCR, and confirm by order-checking.By express the cell of shRNA with the suitable cell preparation of pRS-shRNA plasmid transfection.By suitable marker, select cell as tetracycline, and keep up to colony obvious.Selected clone then, and detect being used to and knock out.

In another embodiment,, express shRNA as pRS-shRNA by plasmid.By knocking out that pRS-shRNA carries out, can be by realizing with Lipofectamine 2000 (Invitrogen) transfectional cell.

In a kind of preferred embodiment, prepare the clone (+/-primary target gene) of coupling by the stable clone of selecting to contain sky pRS carrier or pRS-shRNA.

The shRNA primary target clone's of employing preparation cell carries out the silence of secondary target gene then.Can adopt any known RNA interference method finish the silence of secondary target gene (referring to, for example 5.2 the joint).For example, can be with the plasmid transfection of siRNA and/or coding shRNA, so that the secondary target gene silencing.In one embodiment, the shRNA primary target clone's of preparation cell is dyed in one or more siRNA excess revolutions of deciding the secondary target gene with target.In one embodiment, target is decided one or more siRNA direct transfection of secondary gene in cell.In another embodiment, adopt one or more suitable plasmids, through shRNA target is decided one or more siRNA transfections of secondary gene in cell.Can be after transfection 24 hours results RNA, and knock out by the TaqMan analysis and evaluation.In a kind of preferred embodiment, will contain target and decide the siRNA set of the different siRNA of the kind of k at least of the different sequence areas of secondary target gene (k=2,3,4,5,6 or 10) and be used for the excess revolutions transfect cell.In another kind of preferred embodiment, the siRNA set that will contain the different siRNA of the kind of k at least (k=2,3,4,5,6 or 10) of fixed two or more the different secondary target genes of target is used for transfectional cell.

In a kind of preferred embodiment, total siRNA concentration of the set approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, the total concn of siRNA set is the optimal concentration that is used to make as the target gene silence of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, select the composition of set, the concentration that comprises number with each the different siRNA of different siRNA in the set, make the set of siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another kind of preferred embodiment, the concentration of the every kind of different siRNA that comprises in the set of different siRNA is approximately identical.In another kind of preferred embodiment, the concentration difference each other of the different siRNA that comprises in the set is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a siRNA in the set of different siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of set.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA in the set of different siRNA.Optimal concentration when in other embodiments, the concentration of each siRNA in the set all is lower than independent the use.In a kind of preferred embodiment, the concentration of the siRNA that each in the set is different is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of the different siRNA of each in the set causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the gene silencing that the concentration of every kind of siRNA causes causes when being lower than independent the use, and a large amount of siRNA causes at least 80 or 90% of target gene silence.

In one embodiment, the invention provides and be used to identify the method that shows with collaborative interactional one or more genes that cause death of primary target gene.In the method, the reagent that is used as the inhibitor of the primary target gene in the cell type screens knocking out the library.Therefore, being accredited as the gene of the effect that strengthens reagent, is to have the collaborative interactional gene that causes death with primary target.In a kind of preferred embodiment, described reagent is that target is decided primary target and made its reticent siRNA.

The method of determining the effect of reagent pair cell depends on specific function to be assessed.For example, if reagent is cancer therapy drug, and effect to be assessed is the growth-inhibiting effect of medicine, then can use MTT to measure or alamarBlue measures (referring to, 5.2 joints for example).Those skilled in the art personnel can select method well known in the art based on specific function to be assessed.

In another embodiment, the invention provides and determine the effect of a kind of reagent to the growth of specific cells, the primary target gene of described specific cells and secondary target gene silencing.In a kind of preferred embodiment, according to the clone of preparation mentioned above coupling (+/-primary target gene).Two kinds of clones are dyed in one or more siRNA excess revolutions of deciding the secondary target gene with contrast siRNA (as luciferase) or target then.Under the condition that exposes or be not exposed to described reagent, check the cell cycle spectrum.Can carry out cell cycle analysis (5.2 joints vide infra) with standard method well known in the art.In one embodiment, the supernatant liquor with each hole merges with the cell of gathering in the crops by tryptic digestion.Then with the proper speed centrifugal mixture.Use the 70% ice-cold alcohol time period that cell fixation is suitable then, 30 minutes according to appointment.Can wash the fixed cell once with PBS, resuspended then, for example be resuspended in 0.5ml and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), in suitable temperature, as 37 ℃ of time periods that following incubation is suitable, as 30 minutes.Carry out flow cytometry with flow cytometer.In one embodiment, measure necrocytosis with inferior G1 cell mass.The increase of the G1 cell mass in the cell of primary target gene and secondary target gene silencing shows the collaborative lethal effect of primary and secondary target gene in the presence of described reagent.

In a kind of particular, the invention provides the method that is used for identified gene, knocking out of described gene strengthens the growth-inhibiting effect of KSP inhibitor to tumour cell.In one embodiment, with this method identified gene, knocking out in inferior optimal concentration of described gene promptly is lower than the following growth of tumour cell that suppresses of KSPi existence of the concentration of EC10.In one embodiment, each the siRNA of 3 kinds of siRNA contain in the fixed following 11 kinds of genes of target of preparation and use knocks out the library: CDC20, ROCK2, TTK, FZR1, BUB1, BUB3, BUB1B, MAD1L1, MAD2L1, DNCH1 and STK6 (referring to table 1).KSPi in＜EC10 concentration (25nM), i.e. (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl-2-methyl propylamine (referring to the PCT application PCT/US03/18482 that submitted on June 12nd, 2003) exist or non-existent condition under among these siRNA each is imported HeLa cell, and the reaction of definite cell.The siRNA of a kind of STK6 (STK6-1) shows KSPi and has following remarkable inhibition to growth of tumour cell.

Further check growth inhibitory activity with three kinds of STK6 extra siRNA, and assess all 6 kinds of siRNA and induce the reticent and growth inhibiting ability of STK6.In different siRNA, between reticent level of STK6 and growth-inhibiting, there is good dependency (Fig. 1).This dependency shows that growth-inhibiting is because target activity (that is, STK6 destroys).(1S)-the 1-{[(2S)-4-(2 that in the PCT application PCT/US03/18482 that submitted on June 12nd, 2003, describes then, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl-2-methyl propylamine exist or non-existent condition under, decide contrast (negative control) the titration STK6-1 (Fig. 2) of luciferase with target.The interpolation of KSPi has been moved to the left about 5-10 doubly with the STK6-1 dose response curve.This concentration of KSPi does not have to strengthen the effect of the cell growth that is caused by luciferase siRNA.On the contrary, the dose response curve of target being decided the siRNA of PTEN is not moved by KSPi, and described PTEN has similar effect to the STK-6 cell growth.The siRNA that other target is decided STK6 also strengthens the effect of KSPi cell growth.Thus, the destruction of STK6 has strengthened the effect of KSPi cell growth.The research of the combination of the siRNA by adopting STK6 and KSP has obtained the further support (table 1) to this, and described combination table reveals than arbitrary independent stronger growth inhibitory activity of siRNA.Do not influence cell growth owing to be used for KSPi concentration self of these experiments, active effect shows as collaborative, rather than synergetic KSPi to STK6siRNA.

In another kind of particular, the invention provides the method that is used for determining the collaborative lethal effect between p53 and the CHEK1.The stable clone that has prepared the p53 gene silencing.PRS-TP53 1026 shRNA plasmids are from the library plasmid set deconvolution of TP53, and this is by gathering transform bacteria with this, and seeks the clone who only contains interested plasmid.Employed sequence is as follows: pRS-p53 1,026 19 aggressiveness sequence: GACTCCAGTGGTAATCTAC (SEQ ID NO:43); The primer of sequence-specific PCR: forward: GTAGATTACCACTGGAGTC (SEQ ID NO:44), oppositely: CCCTTGAACCTCCTCGTTCGACC (SEQ ID NO:45).Identify plasmid by sequence-specific PCR, and confirm by order-checking.By with FuGENE 6 (Roche) the transfection HCT116 cell that has the pRS-TP531026 plasmid, prepare stable p 53 clones.After 48 hours cell is assigned in the 10cm culture dish that is added with the 1ug/ml tetracycline, keeps up to colony obvious (5-7 days).The clone is selected 96 orifice plates, in the 1ug/ml tetracycline, keep, and use TP53 and hGUS Pre-Developed to measure reagent (AppliedBiosystems), knock out by the TaqMan check.In order to measure instantaneous knocking out,, after 24 hours, gather in the crops RNA with Lipofectamine 2000 (Invitrogen) transfection HCT116 cell by the pRS-TP531026 plasmid.By TaqMan assessment TP53 transcript level.

Derive from colon tumor cell and be a plurality of tetracycline resistance TP53shRNA clones' (pRS-p53) of HCT116 the target silence (being defined as 50%-96%) of analysis revealed different levels by TaqMan.Fig. 3 shows the TP53 expression level among clone A5 and the A11, and described clone shows the silence of highest level.TP53 silence among these clones has surpassed by transient transfection pRS-p53 to be sent and has been delivered to 24 hours observed silences (Fig. 3) behind the HCT116 cell.Transfection efficiency may the validity of restricted T P53shRNA in transient transfection.Perhaps, the cell with the reticent level of higher TP53 has obtained growth vigor in the clonal growth process.Adopt target to decide the shRNA (pRS-STK6:pRS-STK6 2,178 19 aggressiveness sequence: CATTGGAGTCATAGCATGT (SEQ ID NO:46)) of STK6, also observed the silence of the certain limit in the stable clone.But, these clones do not reach with TP53 clone in the silence of observed the same high level, its silence also is no more than the silence that obtains in instantaneous measurement.This may represent the selection of anti-high-level STK6 silence, because STK6 is the necessary gene of the growth of tumour cell in the culture.

In order to check the TP53 silence among the HCT116 clone whether to compete, dye this clone's cell with the set excess revolutions of CHEK1 specific siRNA with siRNA.The CHK1 set contains following three kinds of siRNA:CUGAAGAAGCAGUCGCAGUTT (SEQ ID NO:99); AUCGAUUCUGCUCCUCUAGTT (SEQID NO:98); And UGCCUGAAAGAGACUUGUGTT (SEQ ID NO:100).Find the reticent activity of the siRNA that the competitive TP53 of reduction of this siRNA set target is fixed.Show siRNA with 10nM or 100nM Oligofectamine (Invitrogen) transfection.For the CHK1 set,, send altogether and pass 100nM with three kinds of siRNA of 33.3nM transfection simultaneously.24 hours results RNA after transfection, and use CHK1 or TP53 and hGUS Pre-Developed to measure reagent (AppliedBiosystems), knock out by the TaqMan analysis and evaluation.Shown in Fig. 4 A, shRNA and siRNA gather the not silence of every kind of other target of competitive inhibition.Measure the inhibition that the known competitive siRNA by the shRNA of the siRNA of the transient transfection of identical sequence or stably express causes then.Shown in Fig. 4 B, three kinds of independent targets are decided KNSL1 (KNSLI210:GACCUGUGCCUUUUAGAGATT (SEQ ID NO:47); KNSLI211:GACUUCAUUGACAGUGGCCTT (SEQ ID NO:48); KNSLI212:AAAGGACAACUGCAGCUACTT (SEQ ID NO:49)) siRNA competitive inhibition is decided the silence (post on the left side) that the siRNA of the cotransfection of STK6 reaches by target.On the contrary, the influence that the silence that homology STK6shRNA causes in the clone of stable transfection is not dyed by the excess revolutions of KNSL1siRNA is even when adding competitor siRNA with high 10 times concentration also be so the post of (middle and the right).These experiments show, almost not competition between the siRNA of the shRNA of stably express and transient transfection.Observations during two kinds of different siRNA of that this competes each other with transfection together, fixed different mRNA of target is opposite, wherein effectively reduces the efficient of one or both siRNA of use.With the siRNA set transient transfection pRS and the pRS-p53HCT116 cell (embodiment 3 vide infra) of about 800 kinds of genes, and the effect of measuring cell growth by Alamar Blue assay method.Observed reaction much at one, do not shown reticent competitive inhibition the set of about 800 kinds of siRNA described in pRS cell and the pRS-p53 cell.

Subsequently, the excess revolutions that assessment CHEK1 siRNA is integrated in the cell of stably express TP53shRNA is dyed, to determine whether to use it for the genetic interaction (SL) between these molecules of research.By select to contain the clone that the A549 lung cancer cell line that free pRS carrier or pRS-p53 are arranged prepares coupling+/-TP53 expresses.A kind of cell in back shows＞90% TP53mRNA silence.Then with contrast luciferase siRNA (luc, 100nM) or CHEK1siRNA set (100nM altogether; Each 33nM of 3 kinds of siRNA) two kinds of clones are dyed in excess revolutions, are exposing or be not exposed to DNA disrupting agent Zorubicin (Dox, their cell cycle spectrum of check under situation Fig. 5).Under the situation that lacks Dox, the cell cycle of pRS-p53 cell spectrum obviously is not different from the cell cycle spectrum of pRS cell.The transient transfection of CHEK1siRNA does not have influence not exist the cell cycle under the Dox situation to compose yet.But under the situation that Dox exists, as desired to the cell of expressing functional TP53, the pRS cells transfected has shown G1 and G2/M stagnates.The excess revolutions of CHEK1siRNA is dyed and has been caused crossing the G2 outpost of the tax office, and increases at the cell number of G1 blocking-up.Because cell keeps the TP53 function, they were stagnated and are not got back to the S phase in the G1 phase.

On the contrary, the pRS-p53 cell has lost the ability of stagnating at G1, and principal reaction handles and stagnate in the G2 phase in Dox, and this is consistent in the effect at the G1 outpost of the tax office with TP53.Cell cycle spectrum by the existing pRS-p53 cell of lucsiRNA excess revolutions hair dyeing does not change (Fig. 5).LucsiRNA even the part that does not cause TP53 to react are recovered (and the corresponding increase at G1 peak), show that this siRNA does not almost have to produce the competitive inhibition of and phenotype reticent to TP53.Therefore, estimate not exist CHEK1 siRNA to gather the competitive inhibition of the TP53 silence that causes.In fact, react on Dox and handle, revealed remarkable change with the pRS-p53 cell of CHEK1 transient transfection at their cell cycle stave, the cell proportion in the S phase significantly increases, and has the DNA that inferior G1 (dead cell) measures.HCT116 cell kind at pRS and pRS-p53 stable transfection has also been observed similar discovery.Therefore, destroying in the time of by the G2 outpost of the tax office of the G1 outpost of the tax office of TP53 mediation and CHEK1 mediation, is lethal to the TP53-tumour cell, but is not lethal to the TP53+ tumour cell.

In another embodiment, the invention provides a member who determines p53 and BRCC mixture, as the method for the collaborative lethal effect between BRCA1, BRCA2, BARD1 and the RAD51.In this embodiment, used a pair of TP53 positive and the negative cells of coupling of decide short hairpin RNA (shRNA) generation of TP53 by the stably express target.Dye TP53 positive or negative cell with the siRNA of BRCA1 or BRCA2 set excess revolutions, use cisplatin treated, and with Alamar Blue analysis of cells grow (Figure 20).When with BRCA1 or BRCA2siRNAs (the about 0.1nM of IC50) transfection, sensitivity was about 10 times when the TP53 negative cells was used contrast siRNA (luciferase, the about 1nM of IC50) transfection to the cis-platinum ratio.In lower cis-platinum concentration, BRCA1 or BRCA2 destroy the back to the sensibilized of cis-platinum even more remarkable.After BRCA1 or BRCA2 destroyed, the TP53 positive cell was to cis-platinum more insensitive (the about 0.4nM of IC50).In this is analyzed, BRCA1 or BRCA2 destroy the back to the sensibilized of cis-platinum on the order of magnitude with sensibilized similar (data not shown goes out) after CHEK1 destruction.Also can use cell cycle analysis, research BRCA1 and BRCA2 destroy the sensibilized of back to the DNA disrupting agent.The TP53 positive and negative cells are dyed in siRNA set excess revolutions with BRCA1 or BRCA2, with a kind of processing in some DNA disrupting agents (cis-platinum, camptothecine, Zorubicin and bleomycin), and by flow cytometry cell cycle destruction.In all cases, the TP53 negative cells BRCA1 and BRCA2 destroy the back than in the luciferase cells transfected to DNA disrupting agent more responsive (data not shown goes out).These cells were shown in Figure 21 to the reaction of bleomycin after BRCA1 destroyed.BRCA1 destroys after handling the TP53 negative cells with bleomycin, has caused more inferior G1 cell (dead cell) than handling the TP53 positive cell.Described result shows that the cell that lacks TP53 destroys the back at BRCA1 and destroys more responsive to DNA.

The clone of using can be positive A549 cell of HeLa cell, TP53 or the negative A549 cell of TP53.In one embodiment, decide the short hairpin RNA (shRNA) of TP53 by the stable transfection target, prepared the positive and negative cells of the TP53 of coupling to (monthlyhighlt highlight, Nov.2003).With the siRNA of 100nM (every kind of siRNA 33nM) set (set of three kinds of siRNA of each gene) transfectional cell, or with the single siRNA transfectional cell of 100nM.Following siRNA:Luc contrast, BRCA1, BRCA2 and BARD1 set have been used.Handle cells transfected with the DNA disrupting agent of various concentration then.The concentration of the every kind of reagent that uses in the cell cycle analysis is as follows: for the HeLa cell, use Zorubicin (10nM), camptothecine (6nM), cis-platinum (400ng/ml), ametycin (40nM), bleomycin (100ng/ml); For other cell, use Zorubicin (200nM), camptothecine (200nM), cis-platinum (2ug/ml) ametycin (400nM), bleomycin (5ug/ml).

In one embodiment, be performed as follows the siRNA transfection: in transfection the day before yesterday, the clone that 2000 (or 100) microlitre is selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) (ATCC Cat.No.CCL-2) is planted in the tissue culturing plate of 6 holes (or 96 holes) with 45,000 (or 2000) cells/well to grow to the about 90% cervical cancer HeLa cell that converges in.For each transfection, with 70 microlitre OptiMEM (Invitrogen) with (Dharmacon, Lafayette CO) mix from 5 microlitre siRNA of 20 micromole's liquid storages.For each transfection, 20 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 25 microlitre OptiMEM/Oligofectamine mixtures are mixed with 75 microlitre OptiMEM/siRNA mixtures, and at room temperature incubation 15-20 minute.100 (or 10) microlitre transfection mixture is distributed in each hole of 6 holes (or 96 holes) plate, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.

After 4 hours, the DMEM/10% foetal calf serum that contains or do not contain the DNA disrupting agent in 100 microlitres/hole is added each hole, to reach the final concentration of every kind of reagent mentioned above.With plate incubation 68 hours again under the condition of 37 ℃ and 5%CO2.Analysis is from the cell cycle spectrum of the sample of 6 orifice plates, with the cell growth of Alamar Blue assay method analysis from the sample of 96 orifice plates.

For cell cycle analysis, will be from the supernatant liquor and the cytomixis of gathering in the crops by tryptic digestion in each hole.Then with 1200rpm with centrifugal 5 minutes of mixture.With 70% ice-cold ethanol with about 30 minutes of cell fixation.With PBS once, be suspended in 0.5ml again and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), and 37 ℃ of following incubations 30 minutes with the fixed cell washing.(Becton Dickinson) carries out flow cytometry with the FACSCalibur flow cytometer, and with FlowJo software (TreeStar, Inc) analytical data.Measure necrocytosis with inferior G1 cell mass.If (siRNA+DMSO) summation of the inferior G1 cell mass of sample and (Luc+ medicine) sample is greater than the inferior G1 cell mass of (siRNA+ medicine) sample, we determine that the siRNA silence is to the sensitization of DNA destructive.

Analyze for Alamar Blue, remove the substratum of 96 orifice plates, add the 100uL/ hole contain 10% (vol/vol) alamar Blue reagent (BioSource International, Inc) and the perfect medium of 1 percent volume 1M Hepes damping fluid tissue culture reagent.Then with plate at 37 ℃ of following incubation 1-4 hours, by exciting and detect emission at 590nm and measure fluorescence at 544nm with SPECTRAMax Gemini-Xs spectrofluorimeter (Molceular Devices).Background (acellular) is proofreaied and correct fluorescent signal.Cell response (survival) under the DNA disrupting agent exists is measured as the per-cent of the control cells growth that does not exist under the DNA disrupting agent condition.

5.2. be used for the method and composition of RNA interference and raji cell assay Raji

The standard method of any gene silencing can be used for the present invention (referring to, for example, Guo etal., 1995, Cell 81:611-620; Fire et al., 1998, Nature 391:806-811; Grant, 1999, Cell 96:303-306; Tabara et al., 1999, Cell 99:123-132; Zamore et al., 2000, Cell 101:25-33; Bass, 2000, Cell 101:235-238; Petcherski et al., 2000, Nature 405:364-368; Elbashir et al., Nature 411:494-498; Paddison et al., Proc.Natl.Acad.Sci.USA 99:1443-1448).Can according to method well known in the art design target decide gene siRNA (referring to, the U.S. Provisional Patent Application No.60/572 of the Jackson that submitted on May 17th, 2004 etc. for example, 314, with Elbashir et al., 2002, Methods 26:199-213, at this complete content of introducing each piece in full as a reference).

Only can use with target gene have the partial sequence homology siRNA (referring to, the international application No.PCT/US2004/015439 of the Jackson that example was submitted on May 17th, 2004 etc. introduces its complete content as a reference in full at this).In one embodiment, use the sense strand continuous nucleotide sequence comprise 11-18 the Nucleotide identical with the sequence of genetic transcription thing siRNA (but this siRNA not with transcript in any sequence have the total length homology) make gene silencing.Preferably, the continuous nucleotide sequence is the central section of siRNA molecule.Continuous nucleotide sequence in the central section can be not since 3 ' any one section successive nucleotide sequence of holding among the siRNA.For example, the continuous nucleotide sequence of 11 Nucleotide can be nucleotide sequence 2-12,3-13,4-14,5-15,6-16,7-17,8-18 or 9-19.In a kind of preferred embodiment, the length of continuous nucleotide sequence is 11-16,11-15,14-15,11,12 or 13 Nucleotide.

In another embodiment, use 3 ' the sense strand continuous nucleotide sequence comprise 9-18 the Nucleotide identical with the sequence of genetic transcription thing siRNA (but this siRNA not with transcript in any continuous sequence have the total length homology) make gene silencing.In this application, the sequence of 3 ' 9-18 Nucleotide is one section successive Nucleotide, and it is since first pair of base, that is, it does not comprise 3 ' overhang of two bases.Therefore, when pointing out that specific nucleotide sequence is positioned at the 3 ' end of siRNA, do not consider 2 base overhangs.In preferred embodiments, the length of continuous nucleotide sequence is 9-16,9-15,9-12,11,10 or 9 Nucleotide.

Can carry out RNA with any method well known in the art disturbs.In one embodiment, by siRNA induced gene silence is provided to cell, the product of simulation nickase cutting (referring to, Elbashir et al. for example, 2001, Nature 411,494-498; Elbashir et al., 2001, Genes Dev.15,188-200 introduces all documents as a reference in full at this).Synthetic siRNA duplex has kept the associating ability with RISC, and instructs the silence of mRNA transcript.SiRNA can chemosynthesis, or derives from the cutting of the double-stranded RNA that is undertaken by the reorganization nickase.Can adopt standard method well known in the art, use the siRNA transfectional cell.

In one embodiment, be performed as follows the siRNA transfection: in transfection the day before yesterday, the cell that 100 microlitres are selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, (ATCC Cat.No.CCL-2) is planted in (Corning in the 96 hole tissue culturing plates with 1500 cells/well to grow to the about 90% cervical cancer HeLa cell that converges in CA), Coming, NY).For each transfection, (Dharmacon Denver) mixes with siRNA from 5 microlitre serial dilutions of 20 micromole's liquid storages with 85 microlitre OptiMEM (Invitrogen).For each transfection, 5 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 10 microlitre OptiMEM/Oligofectamine mixtures and OptiMEM/siRNA mixture are assigned in each test tube, mix, and at room temperature incubation 15-20 minute.10 microlitre transfection mixtures are distributed in each hole of 96 orifice plates, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.

The another kind of method that is used for gene silencing is to import shRNA, promptly short hairpin RNA (referring to, Paddison et al. for example, 2002, Genes Dev.16,948-958; Brummelkampet al., 2002, Science 296,550-553; Sui, G.et al.2002, Proc.Natl.Acad.Sci.USA 99,5515-5520 introduces all above-mentioned documents as a reference in full at this).In the method, from expressing the siRNA that needs as plasmid (or virus) to form hairpin structure with inverted repeats of the ring-shaped sequence that interleaves.The rna transcription thing that contains hairpin structure that obtains by nickase processing is used to carry out reticent siRNA with preparation subsequently.Can in cell, make it possible in vitro and in vivo based on the shRNA of plasmid by stably express, for example in Mammals, carry out long-term gene silencing (referring to, McCaffrey et al.2002, Nature418,38-39; Xia et al., 2002, Nat.Biotech.20,1006-1010; Lewis et al., 2002, Nat.Genetics 32,107-108; Rubinson et al., 2003, Nat.Genetics33,401-406; Tiscornia et al., 2003, Proc.Natl.Acad.Sci.USA 100,1844-1848 introduces all above-mentioned documents as a reference in full at this).Like this, in one embodiment, use shRNA based on plasmid.

In a kind of preferred embodiment, will from the instantaneous importing of the shRNA that recombinant vectors is expressed or stable integration to genome (referring to, Paddison et al. for example, 2002, Genes Dev16:948-958; Sui et al., 2002, Proc Natl Acad Sci USA 99:5515-5520; Yu et al., 2002, Proc Natl Acad Sci U SA 99:6047-6052; Miyagishi etal., 2002, Nat Biotechnol 20:497-500; Paul et al., 2002, Nat Biotechnol20:505-508; Kwak et al., 2003, J Phamacol Sci 93:214-217; Brummelkamp et al., 2002, Science 296:550-553; Boden et al., 2003, Nucleic Acids Res 31:5033-5038; Kawasaki et al., 2003, Nucleic AcidsRes 31:700-707).Can express the siRNA that destroys target gene by the carrier (through shRNA) of any suitable coding shRNA.Carrier also can be encoded and is used to select the marker of specific cloning, and carrier or its enough have been partially integrated in the host genome among the described clone, so that express shRNA.Can carrier be sent with any method well known in the art and be delivered in the cell.In one embodiment, by preparing the cell of expressing shRNA with the suitable cell of plasmid transfection that comprises carrier.Can select cell by suitable marker then.Selected clone then, testing knocks out being used to.In a kind of preferred embodiment, the expression of shRNA is under the control of inducible promoter, can open the silence of its target gene when needing with box lunch.The inducible expression of siRNA is specially adapted to the fixed necessary gene of target.

In one embodiment, the expression of shRNA is under the control of the promotor of being regulated, and described promotor makes it possible to regulate the reticent level of target gene.This makes it possible to screen the cell that target gene is partly knocked out." promotor of being regulated " of Shi Yonging is meant the promotor that can be activated when having suitable inductor herein." inductor " can be anyly can be used to the promotor of being regulated by activation and the molecule of activated transcription.Inductor can be, but be not limited to peptide or polypeptide, hormone or organic molecule.Also can use the analogue of inductor, that is, and the same molecule that activates the promotor of being regulated with inductor.The activity level of the promotor of being regulated by different analogue inductive can be different, make that like this activity level adjusting of the promotor of being regulated is more flexible.The promotor of being regulated in the carrier can be any Mammals transcriptional regulatory well known in the art system (referring to, Gossen et al for example, 1995, Science 268:1766-1769; Lucas et al, 1992, Annu.Rev.Biochem.61:1131; Li et al., 1996, Cell 85:319-329; Saez et al., 2000, Proc.Natl.Acad.Sci.USA97:14512-14517; With Pollock et al., 2000, Proc.Natl.Acad.Sci.USA97:13221-13226).In preferred embodiments, the promotor of being regulated with dosage and/or analogue dependency mode.In one embodiment, by comprising the method for the concentration that the concentration adjustment of inductor is responded to the promotor of being regulated, the activity level of the promotor of being regulated is adjusted to the level that needs.Can be based on the reticent level of the needs of target gene, determine activity level by the needs of using the promotor of being regulated that the specific concentrations inductor obtains.

In one embodiment, use the gene expression system that tsiklomitsin regulates (referring to, Gossen et al for example, 1995, Science 268:1766-1769; U.S. Patent No. 6,004,941).The system that tet regulates uses the composition of procaryotic tet repressor/operon/inductor system to regulate genetic expression in the eukaryotic cell.Like this, the invention provides the expression of using the tet regulation system to regulate the shRNA that is connected with one or more tet operon sequence.This method comprises the carrier transfered cell with the fusion rotein of coding activated transcription.Fusion rotein is included in tsiklomitsin or tetracycline analogue and exists down and tet operon sequence bonded first polypeptide, and described tsiklomitsin or tetracycline analogue are connected with the second polypeptide operability of transcribing in the activating cells.By regulating the concentration of tsiklomitsin or tetracycline analogue, regulate the expression of the shRNA of tet operon connection.

In other embodiments, can use the gene expression system that moulting hormone regulates (referring to, for example, Saez et al., 2000, Proc.Natl.Acad.Sci.USA 97:14512-14517) or the gene expression system regulated of MMTV glucocorticoid responsive element (referring to, for example, Lucas et al, 1992, the Annu.Rev.Biochem.61:1131) expression of adjusting shRNA.

In one embodiment, use coding tetracycline resistance markers and driving pRETRO-SUPER (pRS) carrier from H1 (RNA Pol III) promoter expression shRNA.Can prepare the pRS-shRNA plasmid by standard method well known in the art.In one embodiment, pRS-shRNA is from library plasmid set deconvolution, and this is by gathering transform bacteria with this, and seeks the clone who only contains interested plasmid.Preferably, 19 aggressiveness siRNA sequences are used with the suitable forward and the reverse primer that are used for sequence-specific PCR.Identify plasmid by sequence-specific PCR, and confirm by order-checking.By express the cell of shRNA with the suitable cell preparation of pRS-shRNA plasmid transfection.By suitable marker, select cell as tetracycline, and keep up to colony obvious.Selected clone then, and detect being used to and knock out.In another embodiment,, express shRNA as pRS-shRNA by plasmid.By knocking out that pRS-shRNA carries out, can be by realizing with Lipofectamine 2000 (Invitrogen) transfectional cell.

In another approach, can be delivered to animal with sending in the siRNA body, in the organ or tissue as the people (referring to, Song et al.2003 for example, Nat.Medicine 9,347-351; Sorensenet al., 2003, J.Mol.Biol.327,761-766; Lewis et al., 2002, Nat.Genetics32,107-108 introduces all above-mentioned documents as a reference in full at this).In the method, with the solution intravenous injection of siRNA in animal.SiRNA can arrive purpose organ or tissue then, and effectively reduces the expression of target gene in animal organ or tissue.

Can use any suitable propagation well known in the art or growth-inhibiting to measure the cell growth.In a kind of preferred embodiment, with the MTT proliferation assay (referring to, vande Loosdrechet for example, et al., 1994, J.Immunol.Methods 174:311-320; Ohno et al., 1991, J.Immunol.Methods 145:199-203; Ferrari et al., 1990, J.Immunol.Methods 131:165-172; Alley et al., 1988, CancerRes.48:589-601; Carmichael et al., 1987, Cancer Res.47:936-942; Gerlier et al., 1986, J.Immunol.Methods 65:55-63; Mosmann, 1983, J.Immunological Methods 65:55-63) measures the effect of one or more reagent in cell growth inhibiting.Handle the selected time period of cell with one or more candidate agents of selected concentration, as 4-72 hour.The 3-of cell and appropriate amount (4,5-dimethylthiazole-2-yl)-2 then, 5-phenylbenzene tetrazolium bromide (MTT) be the selected time period of incubation together, as 1-8 hour, makes the cell of survival that MTT is converted into insoluble first

Cell in settling.After removing the excessive MTT that comprises in the supernatant liquor, add suitable MTT solvent, as DMSO solution, with the dissolving first

By determining for example optical density(OD) at 570nm place, measure the proportional MTT concentration of number then with survivaling cell.Can measure the candidate agent of a large amount of different concns, so that determine to cause the concentration of 50% candidate agent that suppresses.

In another kind of preferred embodiment, use the alamarBlue of cell proliferation ^TMMeasure screen can be used for cytostatic one or more candidate agents (referring to, Page etal. for example, 1993, Int.J.Oncol.3:473-476).AlamarBlue ^TMMeasure and measure cellular respiration, and measuring used as viable cell number.The interior environment of proliferative cell has higher reductibility than the interior environment of non-proliferative cell.For example, in breeding, NADPH/NADP, FADH/FAD, the ratio of FMNH/FMN and NADH/NAF increases.These metabolic intermediates can reduce alamarBlue, therefore, can be used to monitor cell proliferation.Cell number in the sample of the processing of measuring by alamarBlue can be expressed as the per-cent with respect to the cell number in the untreated control sample.Can measure the alamarBlue reduction by absorbancy or spectrophotofluorimetry.In one embodiment, determine the alamarBlue reduction, and adopt following formula to be calculated as reduction per-cent by absorbancy:

Wherein:

λ ₁＝570nm

λ ₂＝600nm

(ε _Redλ ₁)=155,677 (molar extinction coefficient of the reductive alamarBlue of 570nm place)

(ε _Redλ ₂)=14,652 (molar extinction coefficient of the reductive alamarBlue of 600nm place)

(ε _Oxλ ₁)=80,586 (molar extinction coefficient of the alamarBlue of 570nm place oxidation)

(ε _Oxλ ₂)=117,216 (molar extinction coefficient of the alamarBlue of 600nm place oxidation)

(A λ ₁The absorbancy of)=570nm place test hole

(A λ ₂The absorbancy of)=600nm place test hole

(A ' λ ₁)=570nm contains at the place that substratum adds alamarBlue but the absorbancy of not adding the negative control hole of cell

(A ' λ ₂)=600nm contains at the place that substratum adds alamarBlue but the absorbancy of not adding the negative control hole of cell.Preferably, deduct the not % reduction in celliferous hole from the % reduction in the hole of containing sample, to determine to be higher than the % reduction of background.

Can adopt standard method well known in the art to carry out cell cycle analysis.In one embodiment, the supernatant liquor with each hole merges with the cell of gathering in the crops by tryptic digestion.Then with the proper speed centrifugal mixture.Use the 70% ice-cold ethanol time period that cell fixation is suitable then, 30 minutes according to appointment.Can wash the fixed cell once with PBS, resuspended then, for example be resuspended in 0.5ml and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), in suitable temperature, as 37 ℃ of time periods that following incubation is suitable, as 30 minutes.Carry out flow cytometry with flow cytometer.In one embodiment, measure necrocytosis with inferior G1 cell mass.For example, if, think that then cell is to the reagent sensitivity with the inferior G1 cell mass of the sample of agent treated inferior G1 cell mass greater than the sample of not using agent treated.

5.3.KSP the purposes of interaction gene and product thereof

The invention provides and utilize and the interactional gene of KSP (" KSP interaction gene "), identify and the albumen of KSP interaction gene or protein-interacting or the method and composition of other molecule as STK6 or TPX2 gene, its product and antibody.In a kind of preferred embodiment, the invention provides STK6 or TPX2 gene as described KSP interaction gene.The present invention also provides and has utilized the KSP interaction gene, screens the interactional method and composition of regulating the expression of KSP interaction gene or regulating KSP interaction gene or albumen and other albumen or molecule as STK6 or TPX2 gene, product and antibody.The present invention also provides and has utilized the KSP interaction gene, screens as STK6 or TPX2 gene, product and antibody can be used for regulating to the Growth Inhibition resistance of KSP inhibitor (KSPi) and/or strengthen the Growth Inhibition method and composition of KSP inhibitor cell or organism.The present invention also provides and has utilized the KSP interaction gene, as STK6 or TPX2 gene, product and antibody diagnose by the mediation of KSP interaction gene to the Growth Inhibition resistance of KSP inhibitor and be used for and use the method and composition of the therapy combination therapy disease of KSP inhibitor.

5.3.1. determine method with KSP interaction gene or the interactional albumen of its product or other molecule

Can use any method that is suitable for detecting protein-protein interaction to identify the KSP interaction protein, as the interaction between STK6 or TPX2 albumen and the another kind of cell protein.Also can adopt method well known in the art to determine the KSP interaction gene, as the interaction between STK6 or TPX2 gene and other cellular elements.

Operable traditional method comprises co-immunoprecipitation, crosslinked and by gradient or chromatography column copurification.Utilization makes it possible to identify and the interactional cell protein of KSP interaction gene product such as these program.In case obtain separating, described cell protein can be identified, can be used in combination with standard technique then, be used for identifying albumen interactional with it.For example, can use technology well known in the art, as the Edman degradation technique (referring to, Creighton for example, 1983, " Proteins:Structures and Molecular Principles ", W.H.Freeman﹠amp; Co., N.Y. pp.34-49) determines at least a portion aminoacid sequence with the interactional cell protein of KSP interaction gene product.Can be with the guidance of the aminoacid sequence that obtains as the oligonucleotide mixture that produces the gene order that can be used to screen the described cell protein of coding.For example, can finish screening by standard hybridization or round pcr.The technology that is used to prepare oligonucleotide mixture and screening be well known in the art (referring to, Ausubel for example, supra., and PCR Protocols:A Guide to Methods and Applications, 1990, Innis, M.et al., eds.Academic Press, Inc., New York).

In addition, can use the gene that causes while identification code and the interactional cell protein of KSP interaction protein.These methods comprise, for example, utilize the similar mode of antibody Detection Techniques with known λ gt11 library, with the KSO interaction protein detection expression library of mark.

Describe the interactional a kind of method in the proteic body that detects in detail, promptly two heterological systems, just in order to illustrate, rather than restriction.Described this system a kind of form (Chien etal., 1991, Proc.Natl.Acad.Sci.USA, 88:9578-9582), and can (Palo Alto CA) is purchased from Clontech.

In brief, adopt such system, made up the plasmid of the two kinds of hybrid proteins of encoding: a kind of DNA by the transcription activating protein that merges with KSP interaction gene product combines the territory and forms, another kind of by with this plasmid of recombinating as the part in cDNA library in the activation domain of the transcription activating protein that merges of the agnoprotein of cDNA coding form.DNA is transformed into the Saccharomyces cerevisiae strain that contains reporter gene (as HBS or lacZ) in conjunction with territory fusion plasmid and cDNA library, and the regulatory region of described yeast strains contains the binding site of transcriptional activator.Any independent hybrid protein all can not activate transcribing of reporter gene: DNA in conjunction with the territory hybrid protein can not because it does not provide mobilizing function, the activation domain hybrid protein can not because it can not navigate to the binding site of activator.Functional activator has been rebuild in the interaction of two kinds of hybrid proteins, and causes the expression of reporter gene, and the mensuration by the reporter gene product can detect it.

Two heterological systems or relevant method can be used for screening and the interactional proteic activation domain of " bait " gene product library.Illustrate, but be not restriction, KSP interaction gene product can be used as the bait gene product.Total genome or cDNA sequence merge with the DNA of coding activation domain.Combine this library of merging in the territory with DNA and reported in the strain to yeast by cotransfection, express those of reporter gene in the transformant that screening obtains with the plasmid of the heterozygote of coding bait KSP interaction gene product.For example, but be not restriction, bait KSP interaction gene sequence as the encoding sequence of KSP interaction gene, can be cloned in the carrier, makes its translation be blended in the DNA of the proteic DNA of coding GAL4 in conjunction with the territory.These bacterium colonies of purifying separate and are responsible for the library plasmid that reporter gene is expressed.Identify by the plasmid-encoded albumen in library with dna sequencing then.

The cDNA library that can prepare clone with the conventional method of implementing in this area, from this library with the interactional albumen of bait KSP interaction gene product be to be detected.According to particular system described herein, for example, the cDNA fragment can be inserted carrier, make their translations be blended in the transcription activating domain of GAL4.This library can with bait KSP interaction gene-GAL4 fusion plasmid cotransformation in yeast strains, this yeast strains contains the lacZ gene by the promoters driven that comprises the GAL4 activation sequence.Interactional with bait KSP interaction gene product, by the albumen that is blended in the GAL4 transcription activating domain of cDNA coding, with the active GAL4 albumen of reconstruct, and therefore drive the HIS3 expression of gene.Can detect the bacterium colony of expressing HIS3 by containing based on the growth on the Petri dish of the substratum of the shortage Histidine of semi-solid agar.Then can be from these bacterial strain purifying cDNA, and with the conventional technology production of implementing in this area with separate bait KSP interaction gene-interaction protein.

Can determine interaction between KSP interaction gene and the conditioning agent thereof by standard method well known in the art.

5.3.2. the method for screening reagent

The invention provides screening and regulate the KSP interaction protein, as the expression of STK6 or TPX2 or regulate itself and other albumen or the interactional method of molecule.

5.3.2.1. screening assay

Designed following mensuration, to identify some compounds, it is incorporated into KSP interaction gene or gene product, is incorporated into interactional other cell protein of KSP interaction gene product, is incorporated into the cellular component that influenced by KSP interaction gene product, as albumen or be incorporated into the compound of the activity (that is, regulating the activity level of STK6 or TPX2 gene expression dose and/or adjusting STK6 or TPX2 gene product) of the interactional compound that disturbs KSP interaction gene or gene product and other cell protein and adjusting KSP interaction gene.Can also utilize the mensuration of identifying the compound that is incorporated into KSP interaction gene adjusting sequence (as promoter sequence), referring to, Platt for example, K.A., 1994, J.Biol.Chem.269:28558-28562 is incorporated herein by reference in full at this, and described compound can be regulated the expression level of KSP interaction gene.Described compound can include, but not limited to influence the little organic molecule of other expression of gene that KSP interaction gene or some participations comprise the approach of KSP interaction gene, or other cell protein.The method of identifying described cell protein is described in above 5.3.1 joint.Described cell protein can participate in the Growth Inhibition of KSP inhibitor and regulate.In addition, in these compounds, comprised the expression that influences the KSP interaction gene and/or the activity of its gene product, and can be used to regulate compound the Growth Inhibition resistance of KSP inhibitor.

Described compound can include, but not limited to peptide, as soluble peptide, includes, but not limited to the member of the fusogenic peptide and the random peptide library of Ig tail; (referring to, Lam for example, K.S.et al., 1991, Nature 354:82-84; Houghten, R.et al., 1991, Nature 354:84-86), and combinatorial chemistry deutero-molecular library, described library by D-and/or L-configuration amino acid form, phospho-peptide (comprises, but be not limited at random or the phospho-peptide library part degeneracy, directed, referring to, Songyang for example, Z.etal., 1993, Cell 72:767-778), antibody (includes, but are not limited to polyclone, mono-clonal, humanization, antiidiotype, chimeric or single-chain antibody and FAb, F (ab ') ₂With FAb expression library fragment and epi-position binding fragment thereof) and little organic or inorganic molecule.

Through being used for, for example, regulate the biological function of KSP interaction gene product, and be used to improve the Growth Inhibition resistance of KSP inhibitor and/or the growth-inhibiting effect of enhancing KSP inhibitor as mensuration compounds identified described herein.The 5.3.2.2. joint that is determined at hereinafter that is used for the validity of detection compound is discussed.

Can design vitro system, to identify the compound that can be incorporated into KSP interaction gene product of the present invention.Compounds identified can be used for, for example, regulate the activity of wild-type and/or KSP interaction gene product mutant, can be used to illustrate the biological function of KSP interaction gene product, can identify that destroying normal KSP interaction gene product interacts, or use in the screening of the described interactional compound of autoclasia.

The principle that is used for identifying the compound that is incorporated into KSP interaction gene product is included in the reaction mixture that is enough to make KSP interaction gene product and test compounds interaction and bonded condition and these two kinds of compositions of time preparation, forms the mixture that can remove in reaction mixture and/or detect thus.Can adopt multiple mode to carry out described mensuration.For example, a kind of method of measuring comprises KSP interaction gene product or test substances is anchored on the solid phase, and detect the KSP interaction gene product/test compounds mixture that is anchored on the solid phase when reaction finishes.In described method in one embodiment, KSP interaction gene product can be anchored on solid surface, and the direct or indirect mark test compounds of grappling not.

In practice, can easily microtiter plate be used as solid phase.Can be by non-covalent or covalent attachment, the one-tenth of grappling is separation-immobilized.Can finish non-covalent adhering to by also dry with the protein solution bag simply by solid surface.Perhaps, can be with treating the proteic specificity immobilized antibody of fixed, preferred monoclonal antibody anchors to solid surface with albumen.Can prepare surface and storage in advance.

In order to carry out this mensuration, loose composition is added the surface of the bag quilt of the composition that contains grappling.After reaction is finished, remain fixed at the alloy that forms remove under the condition of solid surface unreacted composition (as, by washing).Can adopt number of ways to finish the detection that is anchored on the mixture on the solid surface.When preliminary making during front loose composition, the detection that is fixed on lip-deep mark shows and has formed mixture.When not having the loose composition in preliminary making front, can detect with indirect labelling and be anchored on lip-deep mixture, for example, adopt the specific marker antibody (antibody can carry out direct mark or indirect labelling with the anti-Ig antibody of mark subsequently) of the loose composition in front.

Perhaps, can in liquid phase, react,, and detect mixture from unreacted component separating reaction product; For example, the alloy that forms in the specificity immobilized antibody grappling solution with KSP interaction gene product or test compounds is with the mixture of the specific marker antibody test grappling of other composition of possible mixture.

KSP interaction gene or gene product can be in vivo with one or more cells in or extracellular molecules, as protein-interacting.Described molecule can include, but not limited to nucleic acid molecule and the albumen by the method evaluation of 5.3.1 joint description as mentioned.For the purpose of discussing, described molecule is called " binding partners " at this.The activity that the bonded compound of destruction KSP interaction gene product can be used to regulate KSP interaction gene product.The bonded compound that destroys KSP interaction gene product can be used to regulate the expression of KSP interaction gene, for example passes through the combination of the conditioning agent of adjusting KSP interaction gene.Described compound can include, but are not limited to the peptide of all joints of 5.3.2.1 as mentioned description etc., and it can be near KSP interaction gene product.

Be used for identifying and disturb in KSP interaction gene product and the cell thereof or the ultimate principle of the mensuration system of the interactional compound of extracellular binding partners is included in and is enough to that KSP interaction gene product and binding partners are interacted and bonded condition and time preparation comprise the reaction mixture of these two kinds of compositions, form mixture thus.For the inhibition activity of test compounds, preparation feedback mixture under test compounds existence and non-existent condition.Test compounds can be included in the reaction mixture at first, or can add in the time after adding KSP interaction gene product and binding partners thereof.Do not having under the condition of test compounds or with placebo incubation control reaction mixture.Detect the formation of alloy between KSP interaction protein and the binding partners then.In control reaction, form mixture, but in containing the reaction mixture of test compounds, do not form mixture, show that compound disturbs the interaction between KSP interaction protein and the interactional binding partners.In addition, the mixture in the reaction mixture that contains test compounds and normal KSP interaction protein forms, and also can form with the mixture in the reaction mixture that contains test compounds and sudden change KSP interaction protein to compare.Identify to destroy mutant at needs, but do not destroy under the situation of compound of normal KSP interaction protein, this relatively is important.

Can disturb the interactional compound of KSP interaction gene product and binding partners thereof with heterogeneous or homogeneous phase form.Heterogeneous assays comprises KSP interaction gene product or binding partners is anchored on the solid phase, and detect the mixture that is anchored on the solid phase when reaction finishes.In homogeneous determination, in liquid phase, carry out entire reaction.In any method, can change the order that adds reactant, to obtain different information about the compound of test.For example, can identify by the following method by for example competing the interactional test compounds of disturbing between KSP interaction gene product and the binding partners: in the presence of test substances, react, promptly by before adding KSP interaction protein and interactional binding partners or simultaneously test substances is added reaction mixture.Perhaps,, can detect the test compounds of destroying preformed mixture, as the compound with higher binding constant of one of the composition of replacing mixture by after forming mixture, test compounds being added reaction mixture.Various forms has hereinafter briefly been described.

In the heterogeneous assays system, KSP interaction gene product or interactional binding partners are anchored on the solid phase, and the direct or indirect mark material of grappling not.In practice, can utilize microtiter plate easily.Can be by non-covalent or covalent attachment, substance fixed with grappling.Can finish non-covalent adhering to simply by also dry by solid surface with KSP interaction gene product or binding partners solution bag.Perhaps, can material be anchored to solid surface with the specificity immobilized antibody for the treatment of the fixed material.Can prepare surface and storage in advance.

In order to carry out this mensuration, be with or without the surface that under the condition of test compounds the mating partner of fixed material is exposed to the bag quilt.After reaction is finished, remove unreacted composition (for example, by washing) and remain fixed in the mixture of any formation of solid surface.Can finish the detection of the mixture that is anchored on solid surface with multiple mode.When mark in advance during loose material, the detection that is fixed on lip-deep mark shows and has formed mixture.When not in advance during the loose material of mark, can detect with indirect labelling and be anchored on lip-deep mixture, for example, adopt the specific marker antibody (antibody can carry out direct mark or indirect labelling with the anti-Ig antibody of mark subsequently) of initial loose material.According to the order of adding reacted constituent, can detect and suppress the test compounds that mixture formed or destroyed preformed mixture.

Perhaps, can in liquid phase, react,, and detect mixture from unreacted component separating reaction product existing or not existing under the condition of test compounds; For example, the alloy that forms in the specificity immobilized antibody grappling solution with one of binding constituents is with the mixture of the specific marker antibody test grappling of other mating partner.Equally, according to adding the order of reagent, can identify the test compounds that suppresses mixture formation or destroy preformed mixture to liquid phase.

In a kind of alternate embodiment of the present invention, can use homogeneous determination.In the method, the preformed mixture of preparation KSP interaction protein and interactional binding partners, wherein mark KSP interaction gene product or its binding partners, but because mixture forms, the signal that this mark produces by cancellation (referring to, the U.S. Patent No. 4,109 of Rubenstein for example, 496, it utilizes this method to carry out immunoassay).With the competition of one of material in the preformed mixture and replace the interpolation of the test substances of this material, will cause producing the signal that is higher than background.In this way, can identify the interactional test substances of destruction KSP interaction protein/binding partners.

In a kind of specific embodiment, can prepare KSP interaction gene product, fix to adopt recombinant DNA technology.For example, can adopt fusion vector, as pGEX-5X-l, make the coding region and glutathione-S-transferase (GST) gene fusion of KSP interaction gene, amalgamation mode makes and keeps it in conjunction with activity in the fusion rotein that obtains.Can the interactional binding partners of purifying, and be used to adopt the conventional method of implementing in this area to produce monoclonal antibody.For example, can use radio isotope by the conventional method of implementing in this area ¹²⁵The I traget antibody.In heterogeneous assays, gst fusion protein can be anchored to gsh-sepharose 4B as GST-STK6 or GST-TPX2 fusion rotein.Then, can with allow to take place interact and the bonded mode test compounds exist or non-existent condition under the interactional binding partners of adding.When reaction finishes, can the unconjugated material of flush away, can monoclonal antibody adding system with mark in, and it is combined with the compound composition.Can keep and the associating radioactive amount of gsh-sepharose 4B by measuring, detect the interaction between KSP interaction protein and the interactional binding partners.Test compounds successfully suppresses interactional, and the radioactivity that will cause measuring reduces.

Perhaps, can under the condition that does not have solid gsh-sepharose 4B,, be mixed together in the liquid as GST-STK6 gene fusion albumen and interactional binding partners with fusion rotein.Can or add test compounds in these matter interactions afterwards.Then, this mixture can be added gsh-sepharose 4B, the unconjugated material of flush away.Equally, antibody that can be by adding mark and measurement and the associating radioactivity of pearl detect the interactional inhibition degree of KSP interaction gene product/binding partners.

In another embodiment, can adopt substituting in these two kinds of full-length proteins one or both in conjunction with the peptide fragment in territory and use these technology (is under the proteic situation at binding partners) corresponding to KSP interaction protein and/or interactional binding partners.Can use the method for the conventional any number implemented in this area to identify and the separation and combination site.These methods include, but not limited to the mutagenesis of gene of one of proteins encoded and screening coimmunoprecipitation measure in bonded destroy.Can select then to encode anaphragmic in the gene of second kind of material in the mixture.The proteic separately Gene Sequence Analysis of encoding will disclose corresponding to the proteic zone that participates in the interactional combination.Perhaps, can a kind of albumen be anchored to solid surface with method as described above in this section, and it is also combined with its binding partners interaction, described binding partners has been used proteolytic ferment, as trypsin treatment.After the washing, comprise that small peptide in conjunction with the mark in territory can keep and solid material associates, it can separate and identify by amino acid sequencing.Equally, in case obtained the gene of coding binding partners, can carry out the peptide fragment of through engineering approaches with expressing protein to short gene fragment, it is active to detect described segmental combination then, and purifying or synthetic.

For example, but not restriction, can be according to above description in this section, by preparation GST-STK6 or GST-TPX2 fusion rotein and it is combined with the glutathione agarose pearl and STK6 or TPX2 gene product are anchored to solid material.Can use radio isotope,, and use proteolytic ferment, cut as trypsinase as the interactional binding partners of 35S mark.Cleaved products can be added the GST-STK6 or the GST-TPX2 fusion rotein of grappling then, and make in conjunction with taking place.Behind the unconjugated peptide of flush away, can represent the bond material of the mark in binding partner binds territory by the known method wash-out, purifying, and analysis of amino acid sequence.The peptide that produces evaluation like this be can synthesize, or recombinant DNA technology and suitable facilitation albumen fusion adopted.

5.3.2.2. the Growth Inhibition compound of KSP inhibitor is regulated and/or is strengthened in screening

Can further screen the expression of adjusting KSP interaction gene and/or interactional any reagent of KSP interaction protein, as the adjusting and/or the Growth Inhibition abilities of enhancing KSP inhibitor in cell such as antibody of 5.3.2.1 joint compounds identified, KSP interaction protein.Any suitable propagation well known in the art or growth-inhibiting mensuration can be used for this purpose.In one embodiment, candidate agent and KSP inhibitor are applied to the cell of clone, and definite Growth Inhibition changes.Preferably, determine that with the combination of the candidate agent of different concns and different concns KSPi Growth Inhibition changes, so that determine to cause one or more combinations of the concentration of 50% candidate agent that suppresses and KSPi, that is, and IC50.

In a kind of preferred embodiment, with the MTT proliferation assay (referring to, van deLoosdrechet for example, et al., 1994, J.Immunol.Methods 174:311-320; Ohnoet al., 1991, J.Immunol.Methods 145:199-203; Ferrari et al., 1990, J.Immunol.Methods 131:165-172; Alley et al., 1988, Cancer Res.48:589-601; Carmichael et al., 1987, Cancer Res.47:936-942; Gerlieret al., 1986, J.Immunol.Methods 65:55-63; Mosmann, 1983, J.Immunological Methods 65:55-63) screens with the KSPi combination and be used for cytostatic candidate agent.Candidate agent and KSPi with selected concentration handled cell 4-72 hour.The 3-of cell and appropriate amount (4,5-dimethylthiazole-2-yl)-2 then, 5-phenylbenzene tetrazolium bromide (MTT) incubation 1-8 hour together makes the cell of survival that MTT is converted into insoluble first

Cell in settling.After removing the excessive MTT that comprises in the supernatant liquor, add suitable MTT solvent, as DMSO solution, with the dissolving first By determining the optical density(OD) at 570nm place, measure the proportional MTT concentration of number then with survivaling cell.Can measure the candidate agent of a large amount of different concns, so that determine to cause 50% candidate agent that suppresses and the concentration of KSPi.

In another kind of preferred embodiment, use the alamarBlue of cell proliferation ^TMMeasure screen with the KSPi combination be used for cytostatic candidate agent (referring to, Page etal. for example, 1993, Int.J.Oncol.3:473-476).AlamarBlue ^TMMensuration is described in above 5.2 joints.In a kind of particular, carry out alamarBlue ^TMMeasure, whether decide the transfection titre curve of siRNA of KSP interaction gene owing to select the KSPi of concentration to determine target, as 25nM (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-existence of 2-methyl propylamine and changing.With STK6 siRNA transfectional cell.After the siRNA transfection 4 hours, under the condition that has or do not exist KSPi, add the DMEM/10% foetal calf serum in 100 microlitres/hole, with plate incubation 68 hours under the condition of 37 ℃ and 5%CO2.From the hole, remove substratum, and with containing 10% (vol/vol) alamarBlue ^TMReagent (Biosource International Inc., Camarillo, CA) and the DMEM/10% foetal calf serum (Invitrogen) of the 1M Hepes damping fluid tissue culture reagent (Invitrogen) of 0.001 volume replace.With plate 37 ℃ of incubations 2 hours, adopt then Softmax Pro 3.1.2 software (Molecular Devices) SpectraMaxplus read the plate device (Molecular Devices, Sunnyvale, CA) on, 570 and the 600nm wavelength readings.To there be or do not exist 25nM (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-and 2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-compare with the reduction per-cent in the hole of the STK6siRNA transfection of certain titre and the hole of luciferase siRNA transfection under the condition of 2-methyl propylamine.The % reduction numerical value that when not having KSPi the hole of 0nM luciferase siRNA transfection is calculated is considered to 100%.

5.3.2.3. compounds identified

Compounds identified comprises in this screening has proved that selectivity regulates the compound that the Growth Inhibition ability of KSP inhibitor was expressed and regulated and/or strengthened to KSP interaction gene in the cell.These compounds include, but not limited to siRNA, antisense molecule, ribozyme, triple helical, antibody and peptide molecule, aptamrs and little organic or inorganic molecule.

Compounds identified also comprises the interactional compound of regulating KSP interaction protein and other albumen or molecule in this screening.In one embodiment, compounds identified is to regulate the interactional compound of KSP interaction protein and its interaction mating partner in this screening.In another embodiment, compounds identified is to regulate the interactional compound of KSP interaction gene and transcriptional regulatory agent in this screening.

5.3.3. diagnosis

Several different methods can be used for the assessment of diagnosis and prognostic because the KSP interaction gene, the cell that causes as the adjusting defective of STK6 or TPX2 is to the KSP inhibitor, as (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-the Growth Inhibition resistance of 2-methyl propylamine and be used to identify to have the experimenter who the growth-inhibiting effect of KSP inhibitor is had the tendency of resistance.

In one embodiment, this method comprises determines KSP interaction gene expression level in the cell, and the expression level that wherein is higher than predetermined threshold level shows that cell has the KSPi resistance.Preferably, predetermined threshold level is at least 2 times, 4 times, 8 times or 10 times of normal expression level of KSP interaction gene.In another embodiment, the invention provides the method for the KSPi resistance in the assessment cell, comprise and determine in the cell that by the abundance level of KSP interaction gene encoded protein, the abundance water-glass clear-cells that wherein is higher than predetermined threshold level has the KSPi resistance.In another embodiment, the invention provides the method for the KSPi resistance in the assessment cell, comprise and determine in the mammalian cell that by the activity level of KSP interaction gene encoded protein, the activity level that wherein is higher than predetermined threshold level shows that cell has the KSPi resistance.Preferably, abundance or active predetermined threshold level are the normal abundance of KSP interaction protein or at least 2 times, 4 times, 8 times or 10 times of activity level.

Described method is passable, for example, uses reagent such as KSP interaction gene nucleotide sequence and at the interaction gene product, comprises the antibody of its peptide fragment.Particularly, described reagent can be used for, and for example (1) detects the existence that suddenlys change in the KSP interaction gene, or detects overexpression or the expression deficiency of the mRNA of KSP interaction gene with respect to the normal expression level; (2) detect excess abundance or the abundance deficiency of KSP interaction gene product with respect to KSP interaction protein normal level.

For example, can carry out method described herein by the test kit that use comprises at least a specificity KSP interaction gene nucleic acid described herein or anti-KSP interaction protein antibody reagent, described method can be advantageously used in having illness relevant with the KSP interaction gene or unusual patient with diagnosis in for example clinical the setting.

In order to detect the sudden change in the KSP interaction gene, the cell of any tool nuclear can be used as the initial source of genomic nucleic acids.Expression or KSP interaction gene product in order to detect the KSP interaction gene can use any cell type or the tissue of having expressed the KSP interaction gene.

Detection technique based on nucleic acid is described in hereinafter 5.3.3.1 joint.The peptide detection technique is described in hereinafter 5.3.3.2 joint.

5.3.3.1.KSP the detection that interaction gene is expressed

Can utilize KSP interaction gene in many technology for detection cell or tissues, as the expression of STK6 or TPX2, for example existence of the cell levels of KSP interaction gene transcript and/or sudden change or shortage.Can will be used as the starting point of described determination techniques from the nucleic acid of any tool karyocyte, and can be according to well known to a person skilled in the art that the standard nucleic acid preparation procedure separates.For example, can use the expression level of one or more polynucleotide probes measurements KSP interaction gene of the nucleotide sequence that all comprises in the KSP interaction gene, determine the expression level of KSP interaction gene.In particularly preferred embodiment of the present invention, this method is used for the resistance of diagnosing human cancer to the treatment of employing KSPi.

The hybridization or the amplification assay that DNA can be used for biological sample relate to structure unusual of KSP interaction gene with detection, comprise point mutation, insertion, disappearance and chromosome rearrangement.Described mensuration can include, but not limited to Southern and analyze single-strand conformation polymorphism analysis (SSCP), dna microarray analysis and pcr analysis.

The described diagnostic method that is used to detect KSP interaction gene specific mutant can comprise, for example, contact and incubation comprise the nucleic acid of recombinant DNA molecules, cloned genes or its degeneracy variant, it is available from sample, as patient's sample or other suitable cell source derived from the nucleic acid reagent with one or more marks that comprise recombinant DNA molecules, cloned genes or its degeneracy variant, the conditions favouring of contact and incubation carries out specificity annealing with its complementary sequence in these reagent and KSP interaction gene.Preferably, the length of these nucleic acid reagents is 15-30 Nucleotide at least.Behind the incubation, with all unannealed nucleic acid from nucleic acid: remove the KSP interaction gene molecular hybridization body.Detect exist (if having any described molecule) of the nucleic acid carried out hybridization then.Adopt described detection scheme, the nucleic acid from purpose cell type or tissue can be fixed on the solid support of film for example, or be fixed on the frosting on the surface of microtiter plate for example or polystyrene bead.In this case, behind incubation, can easily the nucleic acid reagent of unannealed mark be removed.With well known to a person skilled in the art standard technique, finished the detection of the KSP interaction gene nucleic acid reagent of remaining, annealed, mark.Can compare with the annealing pattern of estimating from normal KSP interaction gene sequence with nucleic acid reagent annealed KSP interaction gene, so that determine whether to exist the sudden change of KSP interaction gene.

The alternative diagnostic methods that is used for detecting the KSP interaction gene specific nucleic acid molecule in patient's sample or other suitable cell source can comprise their amplification, for example pass through pcr amplification (at Mullis, K.B., 1987, U.S. Patent No. 4,683,202), then with the molecule that well known to a person skilled in the art the technology for detection amplification.Desired those compare under the situation of KSP interaction gene of normal copy if can be with the extension increasing sequence that obtains only contain during with nucleic acid amplification, so that determine whether to exist the sudden change of KSP interaction gene.

In the nucleotide sequence of described hybridization and/or the preferred KSP interaction gene of pcr analysis, comprise those that detect KSP interaction gene splice site sudden change existence.

In addition, can carry out known gene type assay technology, in the KSP interaction gene, carry the individuality of sudden change to identify.Described technology comprises, for example, uses restrictive fragment length polymerphism (RFLPs), and it comprises the sequence variations in one of the recognition site of specificity restriction enzyme of use.

In addition, described the method for the dna polymorphism of analyzing the sudden change that can be used for identifying the KSP interaction gene, described method is utilized the existence of the short polyphone reiterated DNA sequences of different numbers between the restriction enzyme sites.For example Weber (U.S. Patent No. 5,075,217 is introduced the document as a reference in full at this) has described the dna marker thing based on length polymorphism in the short polyphone of (dC-dA) n-(dG-dT) n tumor-necrosis factor glycoproteins block.The equipartition of estimating (dC-dA) n-(dG-dT) n block is 30,000-60,000bp.Closely adjacent marker shows altogether heredity of high frequency on the space, and particularly useful to identifying such as the transgenation of the sudden change in the KSP interaction gene and diagnosis disease and the illness relevant with sudden change in the KSP interaction gene.

Caskey etc. (U.S. Patent No. 5,364,759 is introduced the document as a reference in full at this) have described the DNA spectrum analysis that is used to detect short three and TTTC.This method comprises the extraction target DNA, as the KSP interaction gene, and the DNA that amplification is extracted, and the mark tumor-necrosis factor glycoproteins is to form the genotype collection of illustrative plates of individual DNA.

Also can measure the expression level of KSP interaction gene.For example, can separate and utilize the check of hybridization described above or round pcr known or suspect the cell type or the tissue of expressing K SP interaction gene, as the RNA of the cancer cell type of performance KSPi resistance.Isolated cells can be derived from cell culture or patient.The analysis of taking from the cell of culture can be the part that is used as based on the gene therapy technology of cell, or the check compound is to the steps necessary in the cell assessment of the effect of KSP interaction gene expression.Described analysis can disclose the quantitative and qualitative aspect of the expression pattern of KSP interaction gene, comprises activation or deactivation that the KSP interaction gene is expressed.

In a kind of embodiment of described detection scheme, from purpose RNA molecule synthesis cDNA molecule (as, by being cDNA with RNA molecule reverse transcription).Then the sequence in the cDNA is used as nucleic acid amplification reaction, as the template of uses such as pcr amplification reaction.Be selected from KSP interaction gene nucleic acid reagent as the reverse transcription of this method and the nucleic acid reagent of the synthetic initial reagent (as primer) in the nucleic acid amplification step.The preferred length of described nucleic acid reagent is 9-30 Nucleotide at least.In order to detect amplified production, can carry out nucleic acid amplification with radioactivity or nonradioactive labeling's Nucleotide.Perhaps, can prepare enough amplified productions, make and can pass through to use any suitable nucleic acid staining method, as, product shown by the standard ethidium bromide staining.

In addition, can " original position " carry out described KSP interaction gene and express and measure, that is, directly the tissue slice of the patient tissue that obtains from biopsy thing or excision thing (fixing and/refrigerated) be measured, thereby needn't be carried out nucleic acid purification.Can with the nucleic acid of KSP interaction gene as the probe in the described original position program and/or primer (referring to, Nuovo for example, G.J., 1992, " PCR In Situ Hybridization:Protocols And Applications ", Raven Press, NY).

Perhaps, if can obtain the suitable cell of capacity, can carry out standard Northern and analyze, to determine the mRNA expression level of KSP interaction gene.

Also can express, as the cell levels of KSP interaction transcript and/or the existence or the shortage of sudden change with the KSP interaction gene in the dna microarray technical evaluation cell or tissue.In described technology, one or more polynucleotide probes that all comprise the sequence of KSP interaction gene can be used to monitor the expression of KSP interaction gene.Therefore, the invention provides the dna microarray that comprises polynucleotide probes, described probe comprises the sequence of KSP interaction gene.

Any type of dna microarray technology can be used in combination with the present invention.In one embodiment, by cDNA fragment, be deposited on the suitable surface as the PCR product of full-length cDNA, ESTs etc. with the KSP interaction gene, preparation spot cDNA microarray (referring to, DeRisi et al. for example, 1996, Nature Genetics 14:457-460; Shalon etal., 1996, Genome Res.6:689-645; Schena et al., 1995, Proc.Natl.Acad.Sci.U.S.A.93:10539-11286; With Duggan et al., Nature GeneticsSupplement 21:10-14).In another embodiment, by photoetching technique from the teeth outwards original position synthetic contain with the high density oligonucleotide array of the sequence complementary oligonucleotide of KSP interaction gene (referring to, for example, Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc.Natl.Acad.Sci.U.S.A.91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; McGall et al., 1996, Proc.Natl.Acad.Sci.U.S.A.93:13555-13560; United States Patent(USP) Nos. 5,578,832; 5,556,752; 5,510,270; 5,858,659; With 6,040,138).The microarray technology of this form to detect single nucleotide polymorphism (SNPs) particularly useful (referring to, Hacia et al. for example, 1999, Nat Genet.22:164-7; Wang et al., 1998, Science280:1077-82).In another embodiment, by ink-jet technology from the teeth outwards original position synthetic contain with the high density oligonucleotide array of the sequence complementary oligonucleotide of KSP interaction gene (referring to, Blanchard for example, the open WO 98/41531 of disclosed international patent application on September 24th, 1998; Blanchard et al., 1996, Biosensors andBioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arraysin Genetic Engineering, Vol.20, J.K.Setlow, Ed., Plenum Press, New York at pages 111-123).In another embodiment, the dna microarray that allows to carry out the control of electronics severity can be used in combination with the polynucleotide probes of the sequence that comprises the KSP interaction gene (referring to, for example U.S. Patent No. 5,849, and 486).

5.3.3.2.KSP the detection of interaction gene product

Can be according to description herein, with diagnosis and the prognosis agent of the antibody of the KSP interaction gene product of anti-wild-type or sudden change or its conservative variant or peptide fragment as the KSPi resistance.Described diagnostic method can be used to detect expression level unusual of KSP interaction gene, or time, tissue, cell or the Subcellular Localization of the structure of KSP interaction gene product and/or product is unusual.

Because KSP interaction gene product is intracellular gene product, the illness that antibody described below and method of immunity cause in the adjusting defective of assessment treatment KSP interaction gene has importance in the external application as the effect of proliferative disease.Antibody as mentioned below or antibody fragment can be used for the possible treatment compound of in-vitro screening, to determine their effects to expression of KSP interaction gene and the production of KSP interacting peptide.Can identify the compound that the relevant illness of the interactional adjusting defective of KSP is had beneficial effect, and determine the treatment effective dose.

Also can use for example external method of immunity to assess effectiveness based on the gene therapy of cell to the relevant illness of the adjusting defective of KSP interaction gene.Antibody that can the anti-KSP interacting peptide of external use is determined the KSP interaction gene expression level finished in the cell that produces the KSP interacting peptide genetically engineered.Evidence disclosed herein shows that KSP interaction gene product is a gene product in the cell, and described mensuration is preferably carried out with cell lysate or extract.Described analysis makes it possible to determine to obtain the necessary transformant number of interior curative effect, and the optimized gene replacement scheme.

Tissue to be analyzed or cell type generally include those of known or suspection expressing K SP interaction gene, as, KSPi resistance cancer cell type.The protein separating method of Shi Yonging can be herein, for example, those that describe among Harlow and the Lane (Harlow, E.andLane, D., 1988, " Antibodies:A Laboratory Manual ", Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York), be incorporated herein by reference in full at this.Isolated cells can be derived from cell culture or patient.The analysis of taking from the cell of culture can be used to check the effect of compound to the expression of KSP interaction gene.

The preferred diagnostic method that is used to detect KSP interaction gene product or its conservative variant or peptide fragment can comprise, for example immunoassay, wherein the interaction partners by KSP interaction gene product or its conservative variant or peptide fragment and anti-KSP interaction gene product specific antibody its detect.

For example, antibody or the antibody fragment in conjunction with the KSP interaction protein can be used for the existence of KSP interaction gene product or its conservative variant or peptide fragment is carried out quantitatively or qualitative detection.This can (referring to this joint hereinafter) immunofluorescence technique be finished by for example use and opticmicroscope, flow cytometry or the fluorescently-labeled antibody of fluoroscopic examination bonded.If described KSP interaction gene product is at cell surface expression, described technology is particularly preferred.

Be used for antibody of the present invention (or its fragment) and can additionally carry out the histology use, as be used for immunofluorescence or immunoelectron microscope, be used for the in situ detection of KSP interaction gene product or its conservative variant or peptide fragment.Can be by removing histological specimen from the patient, and, finish in situ detection with its antibody that is applied to mark of the present invention.Preferably cover on the biological sample and administration of antibodies (or fragment) by antibody (or fragment) with mark.By using described program, not only can determine the existence of KSP interaction gene product or its conservative variant or peptide fragment, also can determine its distribution in being examined tissue.Employing the present invention those skilled in the art will readily recognize that, can improve in the multiple Histological method (as dyeing procedure) any one, to finish described in situ detection.

There is incubation sample down in the antibody that the immunoassay of KSP interaction gene product or its conservative variant or peptide fragment are usually included in the detectability mark that can identify KSP interaction gene product or its conservative variant or peptide fragment, as biofluid, tissue extract, the cell of new results or the lysate of the cell that incubation is crossed in cell culture, and by any technology for detection bonded antibody well known in the art.

Can make biological sample contact and be fixed on solid support or carrier, as nitrocellulose or other can fixed cell, on the solid support of cell granulations or soluble protein.Can handle with the KSP interaction protein specific antibody of detectability mark subsequently with suitable damping fluid washing upholder then.Can wash solid support to remove unconjugated antibody with damping fluid for the second time then.Can detect the amount of the bonded marker on the solid support then by ordinary method.

" solid support or carrier " is intended to represent can conjugated antigen or any upholder of antibody.Known upholder or carrier comprise glass, polystyrene, polypropylene glycol, polyoxyethylene glycol, dextran, nylon, amylase, natural and Mierocrystalline cellulose, polyacrylamide, gabbro and the magnetite modified.The character of carrier can be soluble to a certain degree or insoluble, to be used for purpose of the present invention.Support material can have any possible node configuration substantially, as long as the link coupled molecule can conjugated antigen or antibody.Therefore, the upholder configuration can be a spheric, as pearl or cylindrical, as is positioned at the outside surface of test trough internal surface or bar.Perhaps, the surface can be flat, as sheet, test strip etc.Preferred upholder comprises polystyrene bead.Those skilled in the art will know that to be used for binding antibody or antigenic many other suitable carriers, maybe can determine by using normal experiment.

Can determine the combination activity of given batch anti-KSP interaction gene product antibody according to known method.Those skilled in the art can determine each operating and optimum condition determination of measuring by normal experiment.

One of approach that can detectability mark KSP interaction gene peptide specific antibody is by it is connected with enzyme, and be used for enzyme immunoassay (EIA) (Voller, A., " TheEnzyme Linked Immunosorbent Assay (ELISA) ", 1978, DiagnosticHorizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A.et al., 1978, J.Clin.Pathol.31:507-520; Butler, J.E., 1981, Meth.Enzymol.73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL; Ishikawa, E.etal., (eds.), and 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo).With the enzyme of antibodies will with suitable substrate, preferred chromophoric substrate reaction, reactive mode makes that generation can be by spectrophotometric for example, fluorescent method or the chemical part by the detection of visual inspection method.The enzyme that can be used to detection property traget antibody comprises, but be not limited to malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, Δ-5-steroidal class isomerase, Alcohol Dehydrogenase from Yeast, alpha-phosphate glyceryl ester, desaturase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase enzymes, rnase, urease, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.The colorimetry of chromophoric substrate that can be by using enzyme is finished detection.Also can finish this detection by the enzymatic reaction degree of naked eyes comparison to the substrate of the standard substance of similar preparation.

Also can finish detection with in multiple other immunoassay any one.For example, by radiolabelled antibody or antibody fragment, can by use radioimmunoassay (RIA) detect the KSP interacting peptide (referring to, Weintraub for example, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March 1986, are hereby incorporated by).Can be by such as using gamma counter or scintillometer or by autoradiographic method detection of radioactive isotropic substance.

Also can use the fluorescent chemicals traget antibody.When fluorescently-labeled antibody is exposed to the light time with suitable wavelength, then because fluorescence can detect its existence.In most of normally used fluorescently-labeled compounds, comprise fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, Phthalyldicarboxaldehyde and fluorescamine.

Also can use the fluorescent emission metal, as ¹⁵²Eu or other lanthanide series metal detectability traget antibody.Can use metal-chelating group that these metals are connected with antibody such as Diethylenetriamine valeric acid (DTPA) or ethylenediamine tetraacetic acid (EDTA) (EDTA).

Also can be by antibody and chemiluminescence compound coupling are detected antibody.Determine the existence of the antibody of chemiluminescent labeling then by the existence that detects the fluorescence that in chemical reaction process, produces.The example of useful especially chemiluminescent labeling compound is luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, heromatic acridinium ester, imidazoles, acridinium salt and barkite.

Equally, can use bioluminescent compound mark antibody of the present invention.Noclilucence is a class chemoluminescence of finding in the biosystem, and wherein catalytic protein has increased the efficient of chemiluminescence reaction.By detecting luminous existence, determine the existence of bioluminescent protein.The important biomolecule luminophor that is used for the mark purpose is fluorescein, luciferase and aequorin.

5.3.4. regulate the method that the KSP interaction gene is expressed

Can use and regulate the KSP interaction gene in the multiple therapy methods body, as the expression of STK6 or TPX2 according to the present invention.For example, can be to the siRNA molecular engineeringization, and be used for making in vivo KSP interaction gene silence.Also can carry out through engineering approaches, and be used for the translation of blocking-up KSP interaction RNA in the body the antisense DNA molecule.Perhaps, can design ribozyme molecule, with cutting in the body and destruction KSP interaction mRNA.In another kind of scheme, design oligonucleotides is so that hybridize with 5 ' district (district that comprises the encoding sequence upstream) of KSP interaction gene, and formation can be used to block or reduce the triple-helix structure that the KSP interaction gene is transcribed.If desired, also can design oligonucleotides so that with the binding site hybridization of negative conditioning agent with form triple-helix structure, so that the combining and strengthen transcribing of KSP interaction gene of blocking-up and negative conditioning agent.

In a kind of preferred embodiment, design siRNA, antisense molecule, ribozyme and triple helical Nucleotide are with the translation that suppresses one or more KSP interaction protein isoforms or transcribe, and may be minimum with other expression of gene influence of the total one or more motifs of KSP interaction gene to other.For reaching this purpose, should design employed oligonucleotide based on the distinctive correlated series of KSP interaction gene.

For example, but be not restriction, oligonucleotide should not drop on the highest zone of nucleotide sequence homology of nucleotide sequence and its gene of KSP interaction gene.Under the situation of antisense molecule, described sequence preference is selected from tabulation above.The length of sequence is at least 18 Nucleotide preferably, so that the enough strong annealing of realization and said target mrna sequence, thereby the translation of prevention sequence.Izant?et?al.，1984，Cell，36：1007-1015；Rosenberg?et?al.，1985，Nature，313：703-706。

Under the situation of " tup " type ribozyme, the target sequence of ribozyme is preferably selected from tabulation above.Ribozyme is the active RNA molecule of endonuclease with high special.Hammerhead ribozyme comprises at least a portion complementary hybridization region of nucleotide sequence and target RNA and is fit to the catalytic domain of cutting target RNA.Hybridization region comprises nine (9) individual or more a plurality of Nucleotide.Therefore, hammerhead ribozyme of the present invention has and sequence complementary hybridization region listed above, and length is at least 9 Nucleotide.The structure of described ribozyme and production are well known in the art, and are described in Haseloff et al., 1988, Nature, 334:585-591 more completely.

Ribozyme of the present invention also comprises RNA endonuclease (after this being called " Cech type ribozyme "), as natural existence the in Tetrahymena Thermophila (being called IVS or L-19IVS RNA), and the ribozyme (Zaug that is fully described by Thomas Cech and co-worker thereof, et al., 1984, Science, 224:574-578:Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; The disclosed international patent application No.WO88/04300 of UniversityPatents Inc.; Been et al., 1986, Cell, 47:207-216).The Cech endonuclease has 8 base pair avtive spots with target RNA sequence hybridization, and the cutting of target RNA is taking place thereafter.

For with 5 ' terminal hybridization of KSP interaction gene and with its formation triple-helix structure, and can be used to block the oligonucleotide of transcribing, they preferably with impregnable other gene of expression level in 5 ' terminal sequence complementation of non-existent KSP interaction gene.Described sequence preferably do not comprise in the promotor of KSP interaction gene yet in addition slightly with the zone of described other dna homolog.Can use aforesaid compound by several different methods well known in the art, include, but not limited to use liposome as delivery vectors.When being in anti-degraded form, also can use naked DNA or RNA molecule, described anti-degraded is by modifying end, by forming ring molecule, or by using alternating bond, comprises the key that phosphorothioate bond and thiophosphoryl are modified.In addition, can pass nucleic acid, wherein nucleic acid molecule be puted together in polylysine or Transferrins,iron complexes by promoting transhipment to send.Also can pass through multiple virus vector, include, but are not limited in retrovirus, vaccinia virus, AAV and the adenovirus any one, with nucleic acid delivery in cell.

Perhaps, can make up coding or as the recombinant nucleic acid molecules of described antisense molecule, ribozyme, triple helical or KSP interaction gene nucleic acid molecule.This nucleic acid molecule can be RNA or DNA.If nucleic acid encoding RNA, this sequence preference is connected with the regulatory element operability, so that produce enough RNA products of the needs of copy.Regulatory element can allow the composing type of sequence or adjustment type to transcribe.Can be in vivo, that is, the transfer vector of one or more RNA that in the cell of biology, will encode, as bacterial plasmid or viral RNA or DNA transfection in cell.(as, Llewellyn et al., 1987, J.Mol.Biol., 195:115-123; Hanahanet al.1983, J.Mol.Biol., 166:557-580).In case be positioned at cell, transfer vector can duplicate, and is transcribed by the cell aggregation enzyme, to produce RNA, perhaps can be integrated into the genome of host cell.Perhaps, can pass through the micromanipulation technology, as micro-injection, the transfer vector transfection of sequence that will comprise one or more RNA that encode makes transfer vector or its be partially integrated in the genome of host cell in cell or transfered cell.

RNAi also can be used to knock out the expression of KSP interaction gene.In one embodiment, be used to the mRNA that degrades with the double stranded rna molecule of 21-23 Nucleotide of the homologous region of the mRNA that transcribes from KSP interaction gene hybridization, thereby make the expression " silence " of KSP interaction gene.Preferably, dsRNA has hybridization region, as, the double stranded region of 19 Nucleotide, and the sequence complementation of the encoding sequence of itself and KSP interaction gene.Any target can be decided the KSP interaction gene, be used for the present invention as the siRNA of the suitable encoding sequence of human STK6 or TPX2 gene.As exemplary embodiment, according to the Standard Selection rule (referring to, Elbashir et al. for example, 2002, Methods 26:199-213 is incorporated herein by reference in full at this) the design target decides the double-stranded siRNA of 21 Nucleotide of the coding region of KSP interaction gene.

Can use any standard method that is used to import siRNA.In one embodiment, the induced gene silence by the siRNA that decides the KSP interaction gene to the presented by cells target (referring to, for example, Elbashir et al., 2001, Nature 411,494-498; Elbashir et al., 2001, Genes Dev.15,188-200 introduces all above-mentioned documents as a reference in full at this).SiRNA can chemosynthesis, perhaps derived from by the cutting of reorganization nickase to double-stranded RNA.The another kind of method that is used to import the double-stranded DNA (dsRNA) that makes KSP interaction gene silence is shRNA, that is, short hairpin RNA (referring to, Paddison et al. for example, 2002, Genes Dev.16,948-958; Brummelkamp et al., 2002, Science296,550-553; Sui, G.et al.2002, Proc.Natl.Acad.Sci.USA 99,5515-5520 introduces all above-mentioned documents as a reference in full at this).In the method, express the siRNA that target decide the KSP interaction gene from plasmid (or virus), it is as inverted repeats, has the ring sequence that interleaves with the formation hairpin structure.The rna transcription thing that contains hair clip that obtains by nickase processing is used for reticent siRNA with generation subsequently.Can be in cell stably express based on the shRNA of plasmid, make and can in cell, carry out long-term gene silencing in vitro and in vivo (referring to, McCaffrey et al.2002, Nature 418,38-39; Xia et al., 2002, Nat.Biotech.20,1006-1010; Lewis et al., 2002, Nat.Genetics 32,107-108; Rubinson et al., 2003, Nat.Genetics 33,401-406; Tiscornia et al., 2003, Proc.AM.Acad.Sci.USA100,1844-1848 introduces all above-mentioned documents as a reference in full at this).Also target can be decided to send in the siRNA body of KSP interaction gene to be delivered to Mammals, in the organ or tissue as the mankind (referring to, Song et al.2003 for example, Nat.Medicine 9,347-351; Sorensen etal., 2003, J.Mol.Biol.327,761-766; Lewis et al., 2002, Nat.Genetics32,107-108 introduces all above-mentioned documents as a reference in full at this).In the method, with the solution intravenous injection of siRNA in Mammals.SiRNA can arrive purpose organ or tissue then, and effectively reduces target gene expression in mammalian organs or the tissue.

5.3.5. regulate the method for active and/or its approach of KSP interaction protein

The activity that can regulate the KSP interaction protein by the interaction of regulating between KSP interaction protein and its binding partners.In one embodiment, can use reagent, as the combination of binding partners as described in antibody, aptamers, the little organic or inorganic molecules in inhibiting, so that regulate the KSPi resistance.In another embodiment, can use reagent, as proteic activity in antibody, aptamers, the little organic or inorganic molecules in inhibiting KSP interaction protein adjusting approach, so that regulate the KSPi resistance.

5.3.6. decide the KSP interaction gene and/or gene product is carried out cancer therapy by target

Can be with adjusting KSP interaction gene mentioned above or albumen, suffer from the patient of cancer as STK6 or TPX2 gene or proteic expression and/or active method and/or composition and KSPi combination therapy.Particularly, described method and/or composition and KSPi can be united use, be used for the treatment of the patient who suffers from the cancer that shows KSP interaction gene or protein mediated KSPi resistance.Described therapy can be used for the treatment of cancer, includes, but not limited to rhabdosarcoma, neuroblastoma and glioblastoma, small cell lung cancer, osteosarcoma, carcinoma of the pancreas, mammary gland and prostate cancer, mouse melanoma and leukemia, and B cell lymphoma.

In preferred embodiments, method of the present invention and/or composition and KSPi unite the patient who is used for the treatment of the cancer of suffering from the KSPi resistance that shows STK6 or TPX2 mediation.In described embodiment, regulate expression and/or the activity of STK6 or TPX2, giving the susceptibility of cancer cells, thereby give or strengthen the validity of KSPi treatment to KSPi.

In combination therapy, can be before using KSPi, simultaneously or use one or more compositions of the present invention afterwards.In one embodiment, before using KSPi, use composition of the present invention.Can determine to use timed interval between composition of the present invention and the KSPi by the normal experiment that those skilled in the art are familiar with.In one embodiment, after reaching the threshold value that needs, KSP interaction protein white level gives KSPi.Can determine the level of KSP interaction protein by adopting above-described any technology.

In another embodiment, use composition of the present invention simultaneously with KSPi.

In another embodiment, also after using KSPi, use one or more compositions of the present invention.When one or more compositions of the present invention that the long half time of KSPi uses in treatment, described using can be useful especially.

It will be understood by those skilled in the art that the arbitrary combination of the different administration time that to use composition of the present invention and KSPi.For example, when the long half time of KSPi during, preferably before or after using KSPi, use composition of the present invention in composition of the present invention.

Use the frequency of composition of the present invention or the KSP interaction protein white level that needs are depended at the interval, can determine this level by above-described any technology.Change into the level that is higher or lower than needs when KSP interaction protein white level, can increase or reduce the frequency of administration of composition of the present invention.

Can by any method assessment well known in the art separately or with the effect or the benefit of the co-administered composition of the present invention of KSPi, for example, by requiring based on the dosage of measuring survival rate, side effect, KSPi or the method for its arbitrary combination.If being applied in of composition of the present invention realized any one or multiple benefit among the patient, as increase survival rate, reduce side effect, reduce the dosage requirement of KSPi, think that then composition of the present invention strengthened the KSPi treatment, and think that this method is effective.

5.3.7. treat cancer by deciding the STK6 gene with the fixed mitotic medication combined target of other target

The contriver finds that also STK6 also decides mitotic other medicines with target, interacts as taxol.Figure 18 shows STK6 makes the Hela cell to the paclitaxel treatment sensitivity.Therefore, the present invention also provides adjusting STK6 mentioned above to express and/or is active so that decide mitotic medicine with target, as taxol combination therapy cancer patients's method and composition.Particularly, described method and/or composition can be united use with taxol, suffer from the patient of the cancer of the taxol resistance that shows the STK6 mediation with treatment.Described therapy can be used for the treatment of cancer, includes, but not limited to rhabdosarcoma, neuroblastoma and glioblastoma, small cell lung cancer, osteosarcoma, carcinoma of the pancreas, mammary gland and prostate cancer, mouse melanoma and leukemia, and B cell lymphoma.

5.4. with the interactional gene of DNA disrupting agent and gene product and uses thereof

The invention provides the method and composition that in the treatment disease, utilizes with interactional gene of DNA disrupting agent and gene product.Described gene is commonly referred to as " DNA destroys response gene ".By the gene product of described genes encoding,, be commonly referred to as " DNA destroys the response gene product " as albumen.The present invention also provides expression/activity of utilizing these genes and product screening regulatory gene/gene product thereof, and/or the interactional method and composition of regulatory gene or albumen and other albumen or molecule.The present invention further provides and utilized these genes and gene product screening to be used for regulating cell the Growth Inhibition susceptibility of DNA disrupting agent and/or the Growth Inhibition compositions and methods and the composition of enhancing cell or biology DNA disrupting agent.The present invention also provides and has utilized the diagnosis of these genes and gene product to the Growth Inhibition resistance or the susceptibility of DNA disrupting agent be used for and adopt the method and composition of the therapy combination therapy disease of one or more DNA disrupting agents.

5.4.1. with interactional gene of DNA disrupting agent and gene product

The invention provides the gene that can weaken or strengthen the cell killing of DNA disrupting agent.These genes can be united use with the DNA disrupting agent of hereinafter 5.4.2 joint description.The purposes of these genes is described in hereinafter 5.4.3 and 5.4.4 joint.

In one embodiment, the invention provides the cell killing that can make the DNA disrupting agent and weaken or strengthen at least 1.5 times, 1.6 times, 1.7 times, 1.8 times and 1.9 gene, as cis, dox or campto.In a kind of preferred embodiment, the invention provides following gene, the cell killing that its silence causes the DNA disrupting agent strengthens at least 2.0 times: BRCA2, EPHB3, WEE1 and ELK1.Fig. 8 has shown that the cell killing that the silence of BRCA2, EPHB3, WEE1 and ELK1 causes the DNA disrupting agent strengthens at least 2 times.The invention provides by uniting adjusting, for example strengthen described expression of gene and/or treat method for cancer by the proteic activity of described genes encoding with comprising the therapy of using the DNA disrupting agent.

The present invention also provides the gene of the cell killing that the DNA disrupting agent that can weaken or strengthen particular type causes.Table II A shows specific gene, and its silence strengthens or weakens the wedding agent by DNA, as DNA ditch wedding agent, as the DNA minor groove binding; The DNA linking agent; Intercalator; Kill with the cell that dna adduct formation agent causes.In one embodiment, the invention provides the gene listed as Table II A, its silence makes the DNA wedding agent, cell killing as cis has strengthened at least 1.5 times, 1.6 times, 1.7 times, 1.8 times and 1.9 times, described gene such as gene I s 752-806 (1.5 times), gene I s 771-806 (1.6 times), gene I s784-806 (1.7 times), gene I s 789-806 (1.8 times) and gene I s 793-806 (1.9 times).In a kind of preferred embodiment, the invention provides following gene, its silence makes the DNA wedding agent, and the cell killing that causes as cis has strengthened at least 2 times: BRCA1, BRCA2, EPHB3, WEE1, ELK1, RPS6KA6, BRAF, GPRK6, MCM3, CDC42, KIF2C, CENPE, CDC25B and C20orf97.In another embodiment, the invention provides following gene, its silence makes the DNA wedding agent, and the cell killing that causes as cis has weakened at least 2 times: PLK (referring to Figure 16).The invention provides by uniting adjusting, for example strengthen or weaken described expression of gene and/or treat method for cancer by the proteic activity of described genes encoding with comprising the therapy of using the DNA disrupting agent.

The present invention also provides and can weaken or strengthen the topoisomerase I inhibitor, the gene of the cell killing that causes as camptothecine.In one embodiment, the invention provides the gene listed as Table II B, its silence makes the topoisomerase I inhibitor, the cell killing that causes as camptothecine has strengthened at least 1.5 times, 1.6 times, 1.7 times, 1.8 times and 1.9 times, described gene such as gene I s 635-807 (1.5 times), gene I s 673-807 (1.6 times), gene I s 702-807 (1.7 times), gene I s 727-807 (1.8 times) and gene I s 749-807 (1.9 times).In a kind of preferred embodiment, the invention provides following gene, its silence makes the topoisomerase I inhibitor, the cell killing that causes as camptothecine has strengthened at least 2 times, described gene such as NM_139286, TOP3B, WASL, STAT4, CHEK1, BCL2, NM_016263, TOP2B, TGFBR1, MAPK8, RHOK, NM_017719, TERT, ANAPC5, NM_021170, SGK2, C20orf97, CSF1R, EGR2, AATK, TCF3, CDC45L, STAT3, PRKY, BMPR1B, KIF2C, PTTG1, NM_019089, FOXO1A, STK4, SRC, ELK1, NM_018492, RASA2, GPRK6, BLK, ABL1, HSPCB, PRKACA, CCNE2, CTNNBIP1, NM_013367, FRAT1, PIK3C2A, NM_017769, XM_170783, NM_016457, XM_064050, STK6, RALBP1, ELK1, NF1, STAT5A, WEE1, PTK6, RPS6KA6, BRCA1, EPHB3 and BRCA2.In another kind of preferred embodiment, the invention provides following gene, its silence makes the topoisomerase I inhibitor, the cell killing that causes as camptothecine has strengthened at least 3 times, described gene such as XM_064050, STK6, RALBP1, ELK1, NF1, STAT5A, WEE1, PTK6, RPS6KA6, BRCA1, EPHB3 and BRCA2.In another embodiment, the invention provides following gene, its silence makes the topoisomerase I inhibitor, the cell killing that causes as camptothecine has weakened at least 2 times, described gene such as PLK, CCNA2, MADH4, NFKB1, RRM2B, TSG101, DCK, CDC5L, CDCA8, NM_006101, INSR.The invention provides by uniting adjusting, for example strengthen or weaken described expression of gene and/or treat method for cancer by the proteic activity of described genes encoding with comprising the therapy of using the topoisomerase I inhibitor.

The present invention also provides and can weaken or strengthen the topoisomerase II inhibitor, the gene of the cell killing that causes as Zorubicin.In one embodiment, the invention provides the gene listed as Table II C, its silence makes the DNA wedding agent, the cell killing that causes as Zorubicin has strengthened at least 1.5 times, 1.6 times, 1.7 times, 1.8 times and 1.9 times, described gene such as gene I s 657-830 (1.5 times), gene I s 685-830 (1.6 times), gene I s 723-830 (1.7 times), gene I s 750-830 (1.8 times) and gene I s 767-830 (1.9 times).In a kind of preferred embodiment, the invention provides following gene, its silence makes the topoisomerase II inhibitor, the cell killing that causes as Zorubicin has strengthened at least 2 times, described gene such as PTK2, KRAS2, BRA, FZD4, RASAL2, CENPE, CCNH, MAP4K3, MAP4K2, ERBB3, RHOK, MYO3A, AXIN1, INPP5D, NM_018401, NEK1, TGFBR1, XM_064050, STAT4, MAP3K1, CCNE2, STK6, HDAC4, CTNNA1, EIF4EBP1, ACVR2B, CDC42, MAPK8, BLK, WEE1, KIF26A, TCF1, NM_019089, NOTCH4, HDAC3, PIK3CB, CCNG2, TLK2, XM_066649, MCM3, ELK1, PTK6, ABL1, FZD4, XM_70783, CHUK, SRC, NM_016263 and C20orf97.In another kind of preferred embodiment, the invention provides following gene, its silence makes the topoisomerase II inhibitor, the cell killing that causes as Zorubicin has strengthened at least 3 times, described gene such as ELK1, PTK6, ABL1, FZD4, XM_170783, CHUK, SRC, NM_016263 and C20orf97.In another embodiment, the invention provides following gene, its silence makes the topoisomerase II inhibitor, and the cell killing that causes as Zorubicin has weakened at least 2 times, described gene such as PLK (referring to Figure 16).The invention provides by uniting adjusting, for example strengthen or weaken described expression of gene and/or treat method for cancer by the proteic activity of described genes encoding with comprising the therapy of using the topoisomerase II inhibitor.

In a kind of preferred embodiment, the invention provides CHEK1, BRCA1, BARD1 and RAD51 as strengthening the gene that kills and wounds of DNA disrupting agent to the p53 cell.

In another kind of preferred embodiment, the invention provides WEE1 as the gene that can weaken or strengthen the cell killing that the DNA disrupting agent causes.Wee1 is the mitotic division negative regulator of at first identifying in fission yeast-grain wine fragmentation sugar yeast (Russell and Nurse, 1987 Cell 49:559-67).The Wee1-mutant has the short G2 phase, and enters mitotic division (so called after wee) when half size of wild-type cell.In the cell of overexpression mitotic division inductor cdc25, the wee1 activity is to prevent that the lethal effect (mitotic division accident) that too early mitotic division causes is necessary.Human homology's thing of wee1 is (Igarashi et al., 1991, the Nature 353:80-83) that clones by the trans-complementation of grain wine fragmentation sugar yeast temperature dependency weel-, cdc25 overexpression mutant.The overexpression of human wee1 has produced the prolongation cell of the inhibition that changes from cell cycle G2-M in the fission yeast.This mankind wee1 clone is significantly less than its yeast counterpart, and finds to lack a part of N-terminal sequence (Watanabe et al., 1995, EMBO 14:1878-91) afterwards.

The human wee1 gene of single copy is positioned on No. 11 karyomit(e) (Taviaux and Demaille, 1993, Genomics 15:194-196).The wee1 gene is 16.96kb, has 11 exons, the mRNA transcript of coding 4.23kb.The human Wee1 albumen of 94kDa comprises 646 amino acid.According to Aceview, that is, and the integrated analysis of public obtainable experimental cDNA database (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi? c=locusid﹠amp; Org=9606﹠amp; 1=7465), may there be 6 kinds of littler wee1 isomer proteins that produce by alternative splicing.Multiple human cell, as lung fibroblast, embryo fibroblast, cervical cancer HeLa cell, adenocarcinoma of colon, bladder cancer (Igarashi et al., 1991, Nature 353:80-83), found the wee1 expression in uterus, blood vessel, liver, eye, spleen, bladder, skin, cartilage and the kinds of tumor cells system (UniGene, http://www.ncbi.nlm.nih.gov/UniGene/).Also found wee1 sample albumen in mouse, rat, beautiful fine rhabditida, fruit bat and Saccharomyces cerevisiae, 646 amino acid whose albumen of mouse and rat have the highest similarity degree (being respectively 89% and 91%) (UniGene).The people Wee1 sequence of total length has 5 sections with the highest PEST scoring, and the catalytic kinase domain is arranged in C-terminal (Watanabe et al., 1995, EMBO 14:1878-91).As if conservative Lys114 residue be crucial (McGowan andRussell, 1993, EMBO 12:75-85) to the weel kinase activity.

In a plurality of species, identified other Wee1 associated kinase.Africa xenopus Wee1 is that parent is expressed (ovocyte), and Wee2 expresses in zygote in non-meristem.In Mammals, relevant Myt1 has the phosphorylation activity similar to Wee1 (summarize in Kellogg, 2003, J.Cell Sci.116:4883-4890).Also identified Wee1B in the mankind, it almost only expresses (Nakanishi et al., 2000, Genes to Cells 5:839-847) in mature oocyte.

Wee1 is the nuclear Tyrosylprotein kinase of a kind of Ser/Thr of belonging to protein kinase family.Cdc-cell periodic protein B kinases when Wee1 changes by the G2/M that suppresses the cell cycle guarantees to finish dna replication dna before mitotic division.The phosphorylation of Thr14 in the ATP-binding site of Cdc2 and Tyr15 residue suppresses its activity; The Wee1 Tyrosylprotein kinase makes the Tyr15 residue phosphorylation of N-terminal.Second kind of relevant protein kinase, promptly Mik1 (Myt1) makes the Cdc2 phosphorylation on Thr14 and the Tyr15.The Cdc2 activity is that to proceed to mitotic division necessary.The dephosphorylation of crucial Tyr15 residue is catalytic by Cdc25, and Cdc25 is opposite with the effect of Wee1.The active balance of Wee1 and Cdc25 determined to enter mitotic division (summarize in Kellogg, 2003, J.Cell Sci.116:4883-4890; Pendergast, 1996, Curr.Opin.Cell Biol.8:174-181).

The Wee1 activity is regulated at the cell cycle camber.In S and G2 phase, Wee1 is active to increase parallel increase protein level.Because excessive phosphorylation and the degraded of Wee1, Wee1 activity are subjected to suppress (Watanabe et al., 1995, EMBO 14:1878-91 when mitotic division; McGowan and Russell, 1993, EMBO 12:75-85).Recently the Cdk1 (Cdc2) that studies have shown that in Africa xenopus and fission yeast can make the wee1 phosphorylation, has shown positive feedback loop model, mitotic division Cdk1 deactivation Wee1 in a small amount wherein, and caused the remarkable increase of mitotic division Cdk1 subsequently.Tome-1 also promotes reduction division to enter by decide Wee1 at G2 phase target to carry out proteolysis destruction by SCF.APC ^CDH1By destroy at G1 Tome-1 and cell periodic protein B make Wee1 recover in the S phase (summarize Surana in Lim and, 2003, Mol.Cell11:845-851).

The new role of Wee1 in apoptosis proposed.The apoptotic Crk that participates in the Africa xenopus can combine with Wee1 by its SH2 structural domain.Exogenous Wee1 with Crk dependency mode quicken the Africa xenopus ovum apoptosis (Smith et al., 2000, J.CellBiol.151:1391-1400).Lacking under the nuclear output factor Crm1 bonded condition, these Crk-Wee1 mixtures also promote in the mammalian cell apoptosis (Smith et al, 2002, Mol.Cell.Biol.22:1412-1423).The research that relates to HIV albumen R (Vpr) also relate to Wee1 in the apoptosis incident (Yuan, et al., 2003, J.Virol.77:2063-2070).Vpr causes the G2 relevant with the Cdc2 deactivation to stagnate, and the G2 that prolongs stagnation causes apoptosis.Wee1 is removed in the apoptosis HeLa of Vpr inductive apoptosis HeLa cell and gammairradiation cell.The overexpression of Wee1 has weakened Vpr inductive apoptosis, and removes by the Wee1 that siRNA causes, and has induced apoptosis death.The mechanism of the obvious conflict of Wee1 level and apoptosis incident and the apoptosis-inducing that undertaken by Wee1 is not also illustrated in these researchs.

If DNA is destroyed, the effect of cell cycle inhibitor is important.Fissional blocking-up has obtained the time that DNA repairs, and make destructive DNA duplicate with separate minimum.Two cell cycles " outpost of the tax office " of genetic integrity are in G1 phase (before DNA is synthetic) and G2 phase (just before mitotic division).Lose these and close card control, promoted cell to evolve and be cancer (summarize Kastan, 1994, Science 266:1821-8) in Hartwell and.

Defective type Wee1 expresses can eliminate the G2 outpost of the tax office, promotes tumor cell proliferation.Have been found that Wee1 significantly is subjected to suppress (summarize in Lee and Yang, 2001, Cell.Mol.Life Sci.58:1907-1922) in colon cancer cell.The shortage that Wee1 expresses also relevant with relatively poor prognosis and higher nonsmall-cell lung cancer recurrence rate (Yoshida et al., 2004, Ann.Onco.15:252-256).

On the contrary, compare with sclerotic tissue on every side, Wee1 level and kinase activity be also rising (Masaki et al., 2003, Hepatology 37:534-543) in hepatocellular carcinoma.

Perhaps, the elimination at the G2 outpost of the tax office can strengthen the chemotherapy to G1 outpost of the tax office defective type tumour cell.A lot of tumour cells lack functional p53 gene, and do not show the G1 outpost of the tax office.Although normal cell will be stagnated at G1 after the DNA that radiotherapy or chemotherapy cause destroys, cancer cells will depend on the G2 outpost of the tax office and carry out DNA and repair.Therefore, the elimination at the G2 outpost of the tax office is more harmful to cancer cells comparison normal cell.Because Wee1 is to the negative adjusting of Cdc2 and weakening of Wee1 pair cell apoptosis, the chemical library screening retrieval that suppresses the compound of Wee1 with selectivity suppresses the carcinostatic agent (Wang et al., 2001, Cancer Res.61:8211-8217) at the G2 outpost of the tax office.The PD0166285Wee1 kinase inhibitor has proved the elimination of stagnating to the inhibition of Cdc2 phosphorylation, to G2 and has made the kill sensitivity of radiotherapy to the p53 mutational cell line.In one embodiment, the invention provides with PD166285 and DNA disrupting agent combination therapy method for cancer.

Wee1 activates the pathology that also may participate in rheumatoid arthritis.Rheumatoid synovial cell growth is phymatoid; Cell has a large amount of tenuigenin, maxicell nuclear and caryogram and changes.These cell transformed are present in the cartilage and bone of human rna and animal model.Rheumatoid synovial cell growth is amorphous and is anchorage independent.C-Fos/Ap-1 transcription factor in the rheumatoid synovial membrane increases.Kawasaki etc. (Kawasaki et al., 2003, Onco.22:6839-6844) proved that Wee1 is by the c-Fos/AP-1 trans-activation; Compare with the osteoarthritis cell, Wee1 significantly increases in the rheumatoid synovial cell.These synovial cells also show the tetraploid character of increase.Deactivation Wee1 can be alleviated some destruction of joint that exist among the RNA.

US20030087847A1 has described with nucleic acid molecule and has suppressed the active method of Chk1, makes the approach of p53 defective type tumour to the chemotherapy sensitivity as eliminating the G2 outpost of the tax office and selectivity.Chk1 makes the inhibition residue phosphorylation on the Cdc25, and Cdc25 is the activator of Cdc2.EP1360281A2 has described Wee1 Nucleotide and aminoacid sequence, the method that the active compound of Wee1 is regulated in the method for express recombinant Wee1 and evaluation.

In another kind of preferred embodiment, the invention provides EPHB3 as the gene that can weaken or strengthen the cell killing that the DNA disrupting agent causes.Receptor tyrosine kinase (RTK) is to have the transmembrane protein of extracellular ligand in conjunction with kinase domain in territory and the cell.Eph acceptor with 14 members comprises maximum RTK subfamily.The outside branch of Eph acceptor comprises immunoglobulin (Ig) (Ig) district (ligand binding domain) of inferring, be to be rich in the zone of halfcystine and near two repetitions of III type fibronectins (Connor and Pasquale, 1995Oncogene 11:2429-2438 the single transmembrane segment subsequently; Labrador etal., 1997, EMBO 16:3889-3897).Tenuigenin partly comprises the tyrosine kinase domain of high conservative, and its flank is lower membrane-proximal region of conservative property and C-terminal tail (sterile α motif and PDZ binding motif).According to the sequence homology of extracellular domain, the Eph acceptor is divided into two groups.The EphA acceptor is with high-affinity and ephrin-A ligand interaction, and this receptor is fixed in cell surface by glycosyl-phosphatidyl inositol (GPI) anchor.The EphB acceptor is preferential in conjunction with striding film ephrin-B part.For each group, acceptor can combine with more than one part, and each part can with more than one receptors bind.Between A and B subgroup, have less receptor-ligand cross-talk and (summarize Klein, 1997Trends in Genetics13:354-359 in Orioli and; Pasquale, 1997Curr.Biol.9:608-615).The Eph acceptor only can activate by the ephrin of film combination or artificial cluster; And the certain bind receptor of soluble ligand, they do not cause acceptor autophosphorylation (Davis et al., 1994Science 266:816-819).Eph acceptor and ephrin are unique, because the two-way signal transmission of they mediations.Because their film bonding state, Eph acceptor and ephrin are considered to mediated cell-cell interaction rather than long scope function.

The Eph receptor expression is uniqueness but eclipsed, shows uniqueness but redundant function.The Eph receptor expression is the highest in nervous tissue, but also is present in many tissues.Be expressed among the embryo of growth higherly, but also be present in the adult tissue.Receptor-ligand binding causes cellular rejection usually, and these repulsive interactions participate in developmental axon guidance, cynapse formation, neural range mode, blood vessel generation and cell migration.The neurocyte that these acceptors also can participate in growing up, blood vessel take place and tumour takes place (to summarize the Pasquale in Dodelet and, 2000Oncogene 19:5614-5619; Zhou, 1998Pharmacol.Ther.77:151-181; Pasquale, 1997Curr.Opin.Cell Biol.9:608-615).Cellular rejection or go to adhere to seemingly and transmit molecule by Eph acceptor and many signals, as the mediation of the interaction between Nck, Ras-GAP, Src, SHEP1 and the SHP2 (Wilkinson, 2001Neurosci.Rev.2:155-164).

There are 8 kinds of EphA acceptors (EphA 1-8) and 6 kinds of EphB (EphB 1-6) acceptor, their about 1000 amino acid whose albumen of all encoding.At many species, as having identified the Eph gene among chicken, rat, mouse and the mankind.EphB3 is also referred to as Hek2, Sek4, Mdk5, Cek10 or Tyro 6, it can with part ephrin-B 1-3 interact (Pasquale, 1997, Curr.Opin.Cell Biol.9:608-615).The EphB3 sequence is high conservative (＞95% amino acid identity) in different plant species.The 20.2kb EphB3 gene of single copy is positioned on human No. 3 karyomit(e)s, has 16 exons.Human protein is formed (ref.seq.NM004443) by 998 amino acid.In embryo development procedure and in adult brain, enteron aisle, placenta, muscle, heart and lung and the kidney (intensity is lower in lung and the kidney), there is high-level mouse EphB3 transcript (Ciossek et al., 1995Oncogene 11:2085-2095).In Adult Human Brain, lung, pancreas, liver, placenta, kidney, skeletal muscle and heart, there is EphB3 transcript (Bohme et al, 1993Oncogene 8:2857-2862).

Identified the EphB3 splice variant in chicken, it has 15 aminoacid insertion (Sajjadi and Pasquale, 1993Oncogene 8:1807-1813) in nearly membrane structure territory.Except main 4.8kb total length EphB3 transcript, littler 2.8kb, 2.3kb and 1.9kb transcript (Ciossek et al., 1995Oncogene 11:2085-2095) in mouse tissue, have been found.In human EphB3, only observed a kind of transcript size (Bohme etal., Oncogene 1993 8:2857-2862).But, identified human EphB2 splice variant, show other isoform (Tang et al., 1998Oncogene 17:521-526) that can find other human Eph acceptor.

Having carried out considerable Eph acceptor in fetal development characterizes.(Genes ﹠amp such as Adams; Dev.13:295-306) proved that EphB3 expresses in yolk sac, and grown the artery and the vein of mice embryonic.They have proved that also the two mutant mices of EphB2/EphB3 show the defective of vitelline vesselization, the expansion of pericardium capsule, vascular development defective and head, heart and body segment blood vessel generation defective.Adams etc. have determined that also the ephrin-B part can induce the capillary vessel in the external test to sprout.

The EphB3 deficient mice has illustrated that acceptor participates in cerebral commissure, particularly connects two Interhemispheric callosal formation.In addition, the EphB2/EphB3 double-mutant has jaw and splits, and shows that they also participate in facial grow (Orioli et al., 1996EMBO 15:6035-6049).

In enteric epithelium, along with differentiation, stem cell produces the precursor with the AD HOC migration.In the intestinal epithelial cells beta-catenin white/sudden change of TCF activates and causes polyp to form.People such as Batle proved beta-catenin white/in the tcf signal transmission incident control colorectal cancer cell and express along the EphB3 of crypts-fine hair axle.In the mouse of no EphB3, migration shows the defective among the sorting cells group to occupy Paneth cell random position in crypts of intestinal crypts bottom usually.In addition, in the EphB2/EphB3 double-mutant, in intestinal epithelial cells, be mixed with the cell (Batle et al., 2002Cell 111:251-263) of propagation and differentiation.

Also in the adult mice cochlea, found the EphB3 expression, shown that it may work in peripheral auditory system.Compare with wild-type contrast, the EphB3 knock-out mice show significantly lower distortion-product ear hearing emission DPOAE level (Howard et al., 2003Hear.Res.178:118-130).The DPOAE observed value has reflected the cochlear function on the external hair cell level.

Willson etc. have proved the rise (Willson et al., 2003, Cell Transpl.12:279-290) that EphB3 expresses in the impaired spinal cord of adult rat damage location.EphB3 receptor expression co is in the CNS zone that also has high-level ephrin B part.The additional expression of damage location EphB3 acceptor and part may be because the damage back suppresses the environment of axon regeneration.

In the tumor cell line of mammary gland and epithelial origin, detected EphB3 (Bohme etal., 1993, Oncogene 8:2857-2862).Also in the kinds of tumors type, raised other Eph receptor expression level (summarizing Pasquale, Oncogene 200019:5614-5619) in Dodelet and.Some evidence promptings, the rise of Eph as if do not drive propagation (Lhotakand Pawson, 1993, Mol.Cell.Biol.13:7071-7079), as if but the expression that raises is relevant (Andres et al., 1994Oncogene 1461-1467 with metastatic potential; Vogtet al., 1998Clin.Cancer Res.4:791-797).

Organizing unordered the adhesion with abnormal cells is the sign of late tumor.It is extremely sensitive that the overexpression of Eph acceptor may make tumour that ephrin is activated, and promotes cell adhesion minimizing, cell movability and invasive.Have been found that the Eph acceptor influences cell-matrix by the adjusting integrin activity and adheres to.Maio et al. (2000Nature Cell.Biol.2:62-69) has proved on the prostate cancer cell ephrin A1 part to the activation of EphA2, the cell attachment of instantaneous inhibition of integrins mediation.In addition, among the Africa xenopus embryo, the ectopic expression of ephrin-Bl or activated EphA4 disturb the cadherin dependent cell to adhere to (Jones et al, 1998Proc.Natl.Acad.Sci.USA 95:576-581 in early days; Winning et al, 1996Dev.Biol., 179:309-319).

Set up the contact between Eph acceptor and the cell skeleton change, this is the ambulant critical aspects of cell.Activate EphB4 by the ephrin-B2 part, induced the film wrinkling (Marston et al., 2003Nat.Cell Biol.5:879-888) of Rac mediation in the Eph express cell.People such as Wahl (2000J.Cell Biol.149:263-270) have proved that ephrin-A5 induces subsiding of nerve growth circular cone in Rho dependency mode.Rho and Rac all with participate in the cell change relevant (summarizing al., 2000Exp.Cell Res.261:1-12) that tumour forms in Schmitz et.The activation of these signal pipelines that the Eph acceptor causes can cause tumour to invade and shift.

Because the effect in embryonic blood vessel growth and blood vessel generation of Eph acceptor and their part (is summarized the Bicknell in Sullivan and, 2003Br.J.Cancer 89:228-231), these molecules also can participate in tumor growth by the vascularization that causes tumour.Verified Eph receptors ligand promotes endothelial cell tissue and is assembled into capillary structure, and induces from the blood vessel that exists and carry out capillary vessel sprout (Daniel et al., 1996Kidney Intl.Suppl.57:S73-81; Pandey et al., 1995Science 268:567-569).But excretory ephrin part also can be used as the chemoattractant of the disperse of endotheliocyte; The eph acceptor of expressing on the tumour cell can instruct from the endotheliocyte that enters and make up neovascularity (Pandey et al., 1995Science 268:567-569).

Since the rise (Bohme et al., 1993Oncogene 8:2857) in tumour cell, and may participate in tumor vessel generation and transfer, and EphB3 can produce the attractive target of cancer diagnosis or treatment interference.Soluble E phA-Fc acceptor suppress the skin window measure in and the tumor vessel that suppresses in the body to have injected in the mouse of 4T1 tumour cell (Brantley et al, 2002 Oncogene 21:7011-7026) take place.

Perhaps, may there be such situation, wherein may needs to strengthen the blood vessel occurrence features of Eph acceptor, for example be used for the treatment of coronary vasodilator and block.

The expression of EphB3 also can be used as the attractive treatment target of CNS damage in the impaired spinal cord.The cellular rejection effect of EphB3 can cause impaired spinal cord aixs cylinder not regenerate.Studies have shown that axon regeneration (Bregman et al., 1995Nature 378:498-501 take place in the impaired spinal cord other minute period of the day from 11 p.m. to 1 a.m when suppress axon regeneration with antibody blocking; GrandPre et al., 2002Nature 417:547-551).

Eph acceptor autophosphorylation be subsequently with have the critical event that signal that Tyrosine O-phosphate combines the SH2 in territory transmits interaction of molecules and (summarize al, 1998Curr.Opion.Neuro.8:375-382) in Bruckner et.

People such as Binns (Binns, et al., 2000, Mol.Cell.Biol.20:4791-4805) describe the ephrin-stimulated cells that is used to study EphB2 on the neuronal cell and measured system.In brief, set up the NG108-15 clone (NG-EphB2WT cell) of stably express EphB2.The feature of NG108-15 cell performance motor neuron, motor neuron are a kind of cell types of expressing EphB2 during fetal development.But, NG108-15 cell not endogenous expression EphB2 or react on the ephrin-B part.Stimulate the NG-EphB2WT cell with Fc-ephrin-B 1, cause spinous process to disappear and the depolymerization of polymerization Actin muscle structure.Expressing cell that tyrosine to phenylalanine replaces (crucial phosphorylation site) in wild-type NG108-15 cell and the nearly film motif does not show the cell bone that reacts on ligand stimulation and reinvents.Also with anti--p Tyr antibody detection wt EphB2 and EphB2 (phosphorylation of tyrosine residues makes a variation in the transformant of Y → F).The EphB2 function of receptors weakens a kind of composition p62 that also causes eph signal transmission cascade ^DokPhosphorylation reduce.

Patent US6169167 has also described with cell-cell autophosphorylation and has measured to determine the hek4 part hek4 activated method.After receptor-ligand binding, from the lysate immunoprecipitation Hek4 acceptor of the Chinese hamster ovary celI of expressing Hek4DNA.Lysate is used to the Western trace that adopts anti-phosphotyrosine antibody to carry out.

In another kind of preferred embodiment, the invention provides RAD51 as the gene that can weaken or strengthen the cell killing of DNA disrupting agent.In mammalian cell, can repair double-stranded DNA fracture (DSBs) by non-homogeneous terminal connection (NHEJ) or by homologous recombination.NHEJ relates to is not having under the situation of template the DNA end to fracture connect again, and can cause the sudden change or the disappearance of broken site.Homologous recombination needs template, complete sister's duplex, and causes the high-fidelity reparation.Homologous recombination is paused in also can DNA plerosis or the replication fork of fracture.The reparation of DSBs is important, because if they are not handled or inaccurate reparation, the impaired or apoptosis of function may take place.Genetic instability is the key feature of tumour cell, and it also can cause not having Hi-Fi homologous recombination reparation.The exchange of the initial step-autosyndetic pairing of homologous recombination and chain, relate to the albumen that belongs to RecA/Rad51 recombinase family (summarize West in Baumann and, 1998, Trends Biochem.Sci.23:247-251; Henning and Sturzbecher, 2003, Toxicology 193:91-109).

E. coli protein RecA is as the conditioning agent to DNA destructive SOS reaction, and promote autosyndetic pairing and chain exchange (summarize West in Baumann and, 1998, TrendsBiochem.Sci.23:247-251).Identified that in Saccharomyces cerevisiae DSB repairs gene rad51, and with recA homology (Shinohara et al., 1992, Cell 69:457-470).Also from human and cloned mouse rad51 gene (Yoshimura et al., 1993, NucleicAcids Res.21:1665; Shinohara et al., 1993, Nature Genet.4:239-243).The human radS1 gene of single copy is positioned at No. 15 karyomit(e)s (Shinohara et al, 1993, Nature Genet.4:239-243).The rad51 gene is made up of 10 exons, 339 the amino acid whose albumen of encoding.Two kinds of proteic aminoacid sequences of Mammals Rad51 and yeast Rad1 have 83% homology, have 56% homology with intestinal bacteria RecA albumen.Homologous region between RecA and the Rad51 comprises and is used to recombinate, UV resistance and oligomer form functional domain (the 321-260 position of RecA) (Yoshimura et al., 1993, Nucleic Acids Res.21:1665; Shinohara et al., 1993, Nature Genet.4:239-243).Finding that mouse Rad51 transcript is high level in thymus gland, spleen, testis and ovary, is low-level (Shinohara et al, 1993, Nature Genet.4:239-243) in brain.Rad51 expresses Cycle Regulation seemingly, transcribes rise (Flygare etal., 1996, Biochim.Biophys.Acta 1312:231-236) during S and G2.In addition, (Rad51B-D), they and Rad51 have 20-30% identity for XRCC2, XRCC3 to have identified 5 kinds of Rad51 parallelism homologues.These parallelism homologues can promote the Rad51 kitchen range form (summarize Schild in Thompson and, 2001, Mutat.Res.477:131-153).

Rad51 works as long spiropolymer, and it twines DNA, to form nucleoprotein filament.Rad51 is incorporated into by the molten nuclearity in DSB site and cuts the single stranded DNA that produces again, and this interaction is strengthened by Rad52.Again Qie Ge DSB end occurs in the Rad51 nucleoprotein filament to the intrusion of homoduplex, need ATP in conjunction with but do not need hydrolysis.Second end that cuts again also caught by Rad51.The end of the cutting again of invading is as DNA synthetic primer again.Holliday-engage untie be connected make the duplex of repairing can separate (summarize in West, 2003, Nat.Rev.Mol.Cell.Biol.4:435-445).People such as Pellegrini (2002, Nature 420:287-293) have reported the motif among the conservative tumor-necrosis factor glycoproteins BRC4 simulation Rad51 among the BRCA2, and as the interface that is used for the monomeric oligomerization of Rad51.Although this BRC4-Rad51 mixture is arranged, BRCA2 can control the assembling of Rad51 nucleoprotein filament.The Rad51 activity also is subjected to the adjusting of other mechanism.Find that the p53 downward modulation is by the promoted homologous recombination of Rad51 (Linke et al., 2003, Cancer Res.63:2596-2605; Yoon et al., 2004, J.Mol.Biol.336:639-654).Find that the Rad51 nucleoprotein filament that Rad54 makes double-stranded DNA (dsDNA) go up formation decomposes, and can participate in the renewal of Rad51-dsDNA silk, this is important in DNA chain permutoid reaction.In yeast, find that Srs2 suppresses reorganization (Veaute et al., 2003, Nature 423:309-312 by the Rad51 silk formation that destroys on the single stranded DNA; Krejci et al., 2003, Nature423:305-309).

Identified the splice variant of Rad51.A kind of transcript (NM_133487) lacks the interior segments corresponding to 5 ' part of exon 4,5 and exon 6, obtains lacking the albumen of 97 amino acid whose interior regions.Transcript by Genbank numbering AY425955 identification also shows the splice variant that has further brachymemma in testis.Also at other species, as found in the caenorhabditis elegant Rad51 splice variant (Rinaldo et al., 1998, Mol.Gen.Genet.260:289-294).

Some studies have shown that Rad51 135C polymorphism significantly raise risk (Levy-Lahad et al., 2001, the Proc.Natl.Acad.Sci.USA 98:3232-3236 of mammary cancer among the carrier of BRCA2 (but not being BRCA1); Kadouri et al., 2004, Br.J.Cancer 90:2002-2005).In suffering from two patients of BILATERAL BREAST CANCER, reported missense mutation (Gln150Arg), but opposite, in most of tumours, do not find Rad51 sudden change (Katoet al., 2000, J.Hum.Genet.45:133-137; Schmutte et al., 1999, CancerRes.59:4564-4569).The Rad51 knock-out mice is dead in early days in fetal development, but heterozygote survival and can educating, and can not set up rad51 ^-/-Mouse cell shows the keying action (Tsuzuki et al., 1996, Proc.Natl.Acad.Sci.USA 93:6236-6240) of this gene.(1998, EMBO J. is 17:598-608) by using the promotor control Rad51 transgenosis that can check to prepare rad51 for people such as Sonoda ^-/-Chicken B lymphocyte DT40 clone.The genetically modified inhibition of rad51 has caused high-caliber rhexis, the cell cycle arrest of G2/M phase and necrocytosis in the DT40 cell.The overexpression of Rad51 in clone also investigated in some researchs.Vispe etc. (1998, Nucleic Acids Res.26:2859-2864) find that the Rad51 overexpression in the Chinese hamster ovary celI has increased the homologous recombination between the homology allelotrope of two adjacency, and have increased late S/G ₂Cell cycle phase is to the resistance of ionizing rays.Work that people such as Richardson (2004, Oncogene 23:546-553) carry out proposed that Rad51 level in the tumour cell increases and the chromosome instability relevant with tumour progression between get in touch.The Rad51 level DSB between inductive phase in mouse ES cells system instantaneous rise 2-4 doubly produced new recombinational repair product and produced abnormal karyotype.

Reported that in tumour the Rad51 level raises, shown that the Rad51 rise can promote tumour progression.People such as Maacke (2000, Int.J.Cancer 88:907-913) have reported the positive correlation between Rad51 overexpression and breast tumor are by stages.Also having observed with non-pernicious control cells in kinds of tumor cells system is that the 2-7 that compares Rad51 doubly increases (Raderschall etal., 2002, Cancer Res.62:219-225).Also in 66% human pancreas adenocarcinoma tissue sample, found the Rad51 overexpression (Maacke et al., 2000, Oncogene19:2791-2795).Rad51 overexpression in the supposition cancer cells can be protected cell to avoid DNA destruction or cause genomic instability and diversity.The expression of also having observed Rad51 in human fibroblast's immortalization raises and recombinates to be increased (Xia et al., 1997, Mol.CellBiol.17:7151-7158).

The function of Rad51 in the tumour resistance pointed out in many researchs.People such as Hansen (2003, Int.J.Cancer 105:472-479) have proved the Etoposide resistance positive correlation in RadS1 level and small cell lung cancer (SCLC) cell.In addition, with the downward modulation of justice or antisense constructs being arranged or raising Rad51, changed the Etoposide susceptibility in the SCLC cell.The treatment of discovery Chlorambucil induces the Rad51 in the B cell chronic lymphocytic leukemia cell to express (Christodoulopoulos et al., 1999, Clin.Cancer Res.5:2178-2184).The antisense Rad51 oligonucleotide DNA that enhanced rad causes in mice embryonic skin cells system and pernicious glioma destroys (Taki et al., 1996, Biochem.Biophys.Res.Commun.223:434-438; Ohnishi et al., 1998, Biochem.Biophys.Res.Commun.245:319-324).The Rad51 downward modulation of carrying out with ribozyme has also increased prostate cancer cell to radiating susceptibility (Collis et al., 2001, Nucleic Acids Res.29:1534-1538).Interaction by the BRC tumor-necrosis factor glycoproteins on Rad51 and the BRCA2 destroys its function, also causes in the cancer cells hypersensitivity (Chen et al., 1999, J.Biol.Chem.274:32931-32935 to radiation and methyl mesylate; Chen et al., 1998, Proc.Natl.Acad.Sci.USA95:5287-5292).People such as Slupianek (2001, Mol.Ceu 8:795-806) have proved that it is important that Rad51 expresses cis-platinum in the myelocyte and ametycin resistance.These research promptings Rad51 improves the attractive target that cancer therapy is renderd a service.

5.4.2.DNA disrupting agent

Can implement the present invention with any known DNA disrupting agent, include, but are not limited to the combination of any topoisomerase enzyme inhibitor, DNA wedding agent, antimetabolite, ionizing rays or two or more described known dna disrupting agents.

The topoisomerase enzyme inhibitor that can unite use with the present invention can be topoisomerase I (TopoI) inhibitor, topoisomerase II (TopoII) inhibitor or dual topoisomerase I and II inhibitor.Topo I inhibitor can be from following any compounds: camptothecin analogues is (as karenitecin, amino camptothecin, lurtotecan, the holder pool is for bearing, irinotecan, BAY 56-3722, rubitecan, GI14721, exatecan mesylate), the rebeccamycin analogue, PNU 166148, rebeccamycin, TAS-103, camptothecine is (as the polyglutamic acid camptothecine, camptothecin sodium), intoplicine, ET 743, J-107088, pibenzimol.The example of preferred topo I inhibitor includes, but not limited to camptothecine, the holder pool replaces and bears (hycaptamine), irinotecan (irinotecan hydrochloride), belotecan, or its analogue or derivative.

Can unite the topoisomerase II inhibitor of use with the present invention can be from following any compounds: anthracycline antibiotic is (as carminomycin, Perarubicin, liposome citric acid daunorubicin, daunomycin, 4-iodo-4-deoxidation Zorubicin.Zorubicin, n, n-phenylbenzene daunomycin, the morpholino Zorubicin, the aclacinomycin microbiotic, duborimycin, menogaril, U-15167, zorubicin, pidorubicin, marcellomycin, detorubicin, annamycin, 7-cyanoquinocarcinol, the deoxidation Zorubicin, darubicin, GPX-100, MEN-10755, valrubicin, KRN5500), epipodophyllotoxin compound is (as resin of podophyllium, teniposide, Etoposide, GL331,2-ethyl hydrazides), the anthrone compound is (as the dihydro-amine anthrone, Bisantrene, mitoxantrone, anthrone), Ciprofloxacin, the carboxamide acridine, amonafide, anthracene pyrazoles microbiotic is (as teloxantrone, sedoxantronetrihydrochloride, piroxantrone, the anthracene pyrazoles, losoxantrone), TAS-103, fostriecin, tetrahydroform, XK469R, XK469, chloroquinoxaline sulfonamide, merbarone, intoplicine, elsamitrucin, CI-921, the pyrazolo acridine, hydroxyl serge carbazole, amsacrine.Preferred topoisomerase II inhibitor comprises, but is not limited to Zorubicin (adriamycin), phosphoric acid Etoposide (etopofos), teniposide, sobuzoxane, or its analogue or derivative.

Can include but not limited to DNA ditch wedding agent with the DNA wedding agent that the present invention unites use, as the DNA minor groove binding; The DNA linking agent; Intercalator; Form agent with dna adduct.The DNA minor groove binding can be an anthracycline antibiotic, the mitomycin microbiotic is (as porfiromycin, KW-2149, Mitomycin B, Mitomycin A, ametycin), chromomycin A3, carzelesin, the actinomycin microbiotic is (as cactinomycin, gengshengmeisu, actinomycin F1), brostallicin, Quinomycin A, bizelesin, duocarmycin microbiotic (as KW 2189), A Duolaixin, the Olivomycine microbiotic, plicamycin, neocarzinostatin, distamycin, MS-247, ET 743, amsacrine, anthramycin and pibenzimol, or its analogue or derivative.

The DNA linking agent includes but not limited to anti-tumor alkylating agent, 8-methoxypsoralen, mitomycin microbiotic, psoralene.Anti-tumor alkylating agent can be that the nitrourea compound is (as cystemustine, tauromustine, Semustine, PCNU, U-9889, SarCNU, CGP-6809, carmustine, Fotemustine, the methyl nitrourea, nimustine, MCNU, the ethyl nitrourea, chlorethyl cyclohexyl nitrosourea, chlorozotocin), mustargen reagent is (as mustard compound, as Spiromustine, Z-4828, Chlorambucil, Emcyt, 2,2, the 2-RA3, prednimustine, Novoembichin, Phenamet, glufosfamide, PTC, ifosfamide, Defosfamide, mustargen, phenesterin, mannomustin, endoxan, melphalan, perfosfamide, Nitromin hydrochloride, uracil mustard, bestrabucil, the DHEA mustargen, tallimustine, mafosfamide, Anilin Mustard, Chlornaphazine; The sulphur mustard compound is as the yperite thing; The mustargen prodrug, as TLK286 and ZD2767), ethylenimine compound is (as the mitomycin microbiotic, ethyleneimine, uredepa, plug is for group, diaziquone, hexamethylene bisacetamide, pentamethylmelamine, hexamethyl melamine, cardinophyllin, triaziquone, tetramethylurethimine, dualar, carboquone), sulfonic acid alkane ester cpds is (as the dimethyl busulfan, Yoshi-864, Improsulfan, piposulfan, treosulfan, busulfan, hepsulfam), epoxide compound is (as anaxirone, mitolactol, dianhydrogalactitol, Teroxirone), the miscellaneous alkylating agent is (as ipomeanol, carzelesin, methylsulfonic acid methylene ester, mitobronitol, bizelesin, U 73975, piperazinedione, VNP40101M, asaley, 6-hydroxymethyl alkyl fulvenes, E09, triethylene glycol diglycidyl ether, ET 743, pipobroman), platinic compound is (as ZD0473, liposome-cis-platinum analogue, satraplatin, BBR 3464, spiroplatin, ormaplatin, cis-platinum, RP-54780, carboplatin, lobaplatin, the folding neratein, iproplatin), triene compound is (as the imidazoles mustargen, CB 10-277, mitozolomide, temozolomide, procarbazine, Dacarbazine), picoline compound (as penclomedine), or its analogue or derivative.The example of preferred alkylating agent includes but not limited to cis-platinum, mitolactol, Fotemustine, ifosfamide, MCNU, nedaplatin (latoplatin), bendamustine (bendamustinehydrochloride), eptaplatin, temozolomide (methazolastone), carboplatin, hexamethyl melamine, prednimustine, RP-54780, carmustine, plug for group, leusulfon (busulfan), lobaplatin, endoxan, bisulfan, alkeran and Chlorambucil, or its analogue or derivative.

Intercalator can be anthrone compound, bleomycin microbiotic, rebeccamycin analogue, acridine, carboxamide acridine, amonafide, rebeccamycin, anthracene pyrazoles microbiotic, Quinomycin A, psoralene, LU 79553, BW A773U, crisnatol mesylate, benzopyrene-7,8-glycol-9,10-epoxide, acodazole, hydroxyl serge carbazole, pixantrone, or its analogue or derivative.

Dna adduct forms agent and includes but not limited to enediyne antitumor antibiotics (as dynemicin A, esperamicin A1, neocarzinostatin, dynemicin, calicheamicingamma II), platinic compound, carmustine, tamoxifen (as 4-hydroxyl-tamoxifen), psoralene, piperazine diaza oxyhydroxide, benzopyrene-7,8-glycol-9,10-epoxide, or its analogue or derivative.

Antimetabolite includes but not limited to cytosine(Cyt), cytosine arabinoside, floxuridine, Fluracil, purinethol, gemcitabine and methotrexate (MTX).

Ionizing rays includes but not limited to X ray, gamma-rays and electron beam.

5.4.3. determine to destroy the method for the interactional albumen of response gene or other molecule with DNA

Can be with being suitable for detecting any method identification of dna destruction reactive protein of protein-protein interaction and the interaction between the another kind of cell protein.Also can be with the interaction between method identification of dna destruction response gene well known in the art and other cellular elements, as the interaction between reaction of DNA destruction and the conditioning agent thereof.

Operable traditional method comprises co-immunoprecipitation, crosslinked and by gradient or chromatography column copurification.Utilization makes it possible to identify with DNA and destroys the interactional cell protein of response gene product such as these program.In case obtain separating, described cell protein can be identified, can be used in combination with standard technique then, be used for identifying albumen interactional with it.For example, can use technology well known in the art, as the Edman degradation technique (referring to, Creighton for example, 1983, " Proteins:Structures and Molecular Principles ", W.H.Freeman﹠amp; Co., N.Y., pp.34-49) definite and DNA destroys at least a portion aminoacid sequence of the interactional cell protein of response gene product.Can be with the guidance of the aminoacid sequence that obtains as the oligonucleotide mixture that produces the gene order that can be used to screen the described cell protein of coding.For example, can finish screening by standard hybridization or round pcr.The technology that is used to prepare oligonucleotide mixture and screening be well known in the art (referring to, Ausubel for example, supra., and PCR Protocols:A Guide to Methods and Applications, 1990, Innis, M.et al., eds.Academic Press, Inc., New York).

In addition, can use the gene that causes while identification code and DNA to destroy the interactional cell protein of reactive protein.These methods comprise, for example, utilize the mode similar to the antibody Detection Techniques in known λ gt11 library to utilize DNA to destroy reactive protein, survey expression library with the DNA destruction reactive protein of mark.

Describe the interactional a kind of method in the proteic body that detects in detail, promptly two heterological systems, just in order to illustrate, rather than restriction.Described this system a kind of form (Chien etal., 1991, Proc.Natl.Acad.Sci.USA, 88:9578-9582), and can (Palo Alto CA) is purchased from Clontech.

In brief, adopt such system, made up the plasmid of the two kinds of hybrid proteins of encoding: a kind ofly form by combining the territory with DNA that DNA destroys the transcription activating protein that the response gene product merges, another kind of by with this plasmid of recombinating as the part in cDNA library in the activation domain of the transcription activating protein that merges of the agnoprotein of cDNA coding form.DNA is transformed into the Saccharomyces cerevisiae strain that contains reporter gene (as HBS or lacZ) in conjunction with territory fusion plasmid and cDNA library, and the regulatory region of described yeast strains contains the binding site of transcriptional activator.Any independent hybrid protein all can not activate transcribing of reporter gene: DNA in conjunction with the territory hybrid protein can not because it does not provide mobilizing function, the activation domain hybrid protein can not because it can not navigate to the binding site of activator.Functional activator has been rebuild in the interaction of two kinds of hybrid proteins, and causes the expression of reporter gene, and the mensuration by the reporter gene product can detect it.

Two heterological systems or relevant method can be used for screening and the interactional proteic activation domain of " bait " gene product library.Illustrate, but be not restriction, DNA destroys the response gene product can be used as the bait gene product.Total genome or cDNA sequence merge with the DNA of coding activation domain.The plasmid that combines the heterozygote that this library of merging in the territory and coding bait DNA destroy the response gene product with DNA in the strain of yeast report, is expressed those of reporter gene by cotransfection in the transformant that screening obtains.For example, but be not restriction, bait DNA destroys the response gene sequence, as the encoding sequence of DNA destruction response gene, can be cloned in the carrier, makes its translation be blended in the DNA of the proteic DNA of coding GAL4 in conjunction with the territory.These bacterium colonies of purifying separate and are responsible for the library plasmid that reporter gene is expressed.Identify by the plasmid-encoded albumen in library with dna sequencing then.

The cDNA library that can prepare clone with the conventional method of implementing in this area, from this library to destroy the interactional albumen of response gene product with DNA be to be detected.According to particular system described herein, for example, the cDNA fragment can be inserted carrier, make their translations be blended in the transcription activating domain of GAL4.Response gene-GAL4 fusion plasmid cotransformation can be destroyed in yeast strains with bait DNA in this library, and this yeast strains contains the lacZ gene by the promoters driven that comprises the GAL4 activation sequence.With DNA destroy the response gene product interactional, by the albumen that is blended in the GAL4 transcription activating domain of cDNA coding, with the active GAL4 albumen of reconstruct, and therefore drive the HIS3 expression of gene.Can detect the bacterium colony of expressing HIS3 by containing based on the growth on the Petri dish of the substratum of the shortage Histidine of semi-solid agar.Then can be from these bacterial strain purifying cDNA, and with the conventional technology production of implementing in this area with separates bait DNA destruction response gene-interaction protein.

Can determine that DNA destroys the interaction between response gene and the conditioning agent thereof by standard method well known in the art.

5.4.4. the method for screening reagent

The invention provides screening regulates the expression of DNA destruction reaction or regulates the interactional method that DNA destroys reaction and other albumen or molecule.

5.4.4.1. screening assay

Designed following mensuration, to identify some compounds, it is incorporated into DNA and destroys response gene or gene product, be incorporated into DNA and destroy interactional other cell protein of response gene product, be incorporated into and be subjected to DNA to destroy the cellular component that the response gene product influences, as albumen, or be incorporated into the interactional compound of interference DNA destruction response gene or gene product and other cell protein and regulate the compound that DNA destroys the activity (that is, regulating the level that DNA destruction response gene expression level and/or adjusting DNA destroy the response gene its lytic activity) of response gene.Can also utilize and identify that being incorporated into DNA destroys the mensuration that response gene is regulated the compound of sequence (as promoter sequence), referring to, Platt for example, K.A., 1994, J.Biol.Chem.269:28558-28562 is incorporated herein by reference in full at this, and described compound can be regulated the expression level that DNA destroys response gene.Described compound can include, but not limited to influence the little organic molecule that DNA destroys other expression of gene of response gene or some participation DNA destruction reaction paths, or other cell protein.The method of identifying described cell protein is described in above 5.4.3 joint.Described cell protein can participate in the Growth Inhibition of DNA disrupting agent and regulate.In addition, in these compounds, comprised that influencing DNA destroys the expression of response gene and/or the activity of DNA destruction response gene product, and can be used to regulate compound the Growth Inhibition resistance of DNA disrupting agent.

Described compound can include, but not limited to peptide, as soluble peptide, includes, but not limited to the member of the fusogenic peptide and the random peptide library of Ig tail; (referring to, Lam for example, K.S.et al., 1991, Nature 354:82-84; Houghten, R.et al., 1991, Nature 354:84-86), and combinatorial chemistry deutero-molecular library, described library by D-and/or L-configuration amino acid form, phospho-peptide (comprises, but be not limited at random or the phospho-peptide library part degeneracy, directed, referring to, Songyang for example, Z.etal., 1993, Cell 72:767-778), antibody (includes, but are not limited to polyclone, mono-clonal, humanization, antiidiotype, chimeric or single-chain antibody and FAb, F (ab ') ₂With FAb expression library fragment and epi-position binding fragment thereof) and little organic or inorganic molecule.

Through being used for, for example, regulate the biological function that DNA destroys the response gene product, and be used to improve the Growth Inhibition resistance of DNA disrupting agent and/or the growth-inhibiting effect of enhancing DNA disrupting agent as mensuration compounds identified described herein.The 5.4.4.2. joint that is determined at hereinafter that is used for the validity of detection compound is discussed.

Vitro system can be designed, the compound that DNA of the present invention destroys the response gene product can be incorporated into to identify.Compounds identified can be used for, for example, the DNA that regulates wild-type and/or sudden change destroys the activity of response gene product, can be used to illustrate the biological function that DNA destroys the response gene product, can identify that destroying normal DNA destroys the interaction of response gene product, or use in the screening of the described interactional compound of autoclasia.

Be used for identifying that being incorporated into principle that DNA destroys the compound of response gene product is included in and is enough to make DNA destroy the response gene product and test compounds interacts and the reaction mixture of bonded condition and these two kinds of compositions of time preparation, form the mixture that in reaction mixture, to remove and/or to detect thus.Can adopt multiple mode to carry out described mensuration.For example, a kind of method of measuring comprises destroys the response gene product with DNA or test substances anchors on the solid phase, and detects the DNA that is anchored on the solid phase and destroy response gene product/test compounds mixture when reaction finishes.In described method in one embodiment, DNA can be destroyed the response gene product and be anchored on solid surface, and the direct or indirect mark test compounds of grappling not.

In practice, can easily microtiter plate be used as solid phase.Can be by non-covalent or covalent attachment, the one-tenth of grappling is separation-immobilized.Can finish non-covalent adhering to by also dry with the protein solution bag simply by solid surface.Perhaps, can be with treating the proteic specificity immobilized antibody of fixed, preferred monoclonal antibody anchors to solid surface with albumen.Can prepare surface and storage in advance.

In order to carry out this mensuration, loose composition is added the surface of the bag quilt of the composition that contains grappling.After reaction is finished, remain fixed at the alloy that forms remove under the condition of solid surface unreacted composition (as, by washing).Can adopt number of ways to finish the detection that is anchored on the mixture on the solid surface.When preliminary making during front loose composition, the detection that is fixed on lip-deep mark shows and has formed mixture.When not having the loose composition in preliminary making front, can detect with indirect labelling and be anchored on lip-deep mixture, for example, adopt the specific marker antibody (antibody can carry out direct mark or indirect labelling with the anti-Ig antibody of mark subsequently) of the loose composition in front.

Perhaps, can in liquid phase, react,, and detect mixture from unreacted component separating reaction product; For example, destroy the alloy that forms in the specificity immobilized antibody grappling solution of response gene product or test compounds, with the mixture of the specific marker antibody test grappling of other composition of possible mixture with DNA.

DNA destroy response gene or gene product can be in vivo with one or more cells in or extracellular molecules, as protein-interacting.Described molecule can include, but not limited to nucleic acid molecule and the albumen by the method evaluation of 5.4.3 joint description as mentioned.For the purpose of discussing, described molecule is called " binding partners " at this.The bonded compound that destroys DNA destruction response gene product can be used to regulate the activity that DNA destroys the response gene product.The bonded compound that destroys DNA destruction response gene product can be used to regulate the expression that DNA destroys response gene, for example by regulating the combination that DNA destroys the conditioning agent of response gene.Described compound can include, but are not limited to the peptide of all joints of 5.4.4.1 as mentioned description etc., and it can destroy the response gene product near DNA.

Be used for identifying and disturb DNA to destroy in response gene product and the cell thereof or the ultimate principle of the mensuration system of the interactional compound of extracellular binding partners is included in and is enough to make DNA destroy the response gene product and binding partners interacts and bonded condition and time preparation comprise the reaction mixture of these two kinds of compositions, form mixture thus.For the inhibition activity of test compounds, preparation feedback mixture under test compounds existence and non-existent condition.Test compounds can be included in the reaction mixture at first, or can add in the time after adding DNA destruction response gene product and binding partners thereof.Do not having under the condition of test compounds or with placebo incubation control reaction mixture.Detect the formation that DNA destroys alloy between response gene albumen and the binding partners then.In control reaction, form mixture, but in containing the reaction mixture of test compounds, do not form mixture, show that compound disturbs the interaction between DNA destruction response gene albumen and the interactional binding partners.In addition, the mixture in containing the proteic reaction mixture of test compounds and normal DNA destruction response gene forms, and also can compare with the mixture formation that contains in the proteic reaction mixture of test compounds and mutant DNA destruction response gene.Identify the destruction mutant at needs, but do not destroy under the situation of the proteic compound of normal DNA destruction response gene that this relatively is important.

Can disturb DNA to destroy the interactional compound of response gene product and binding partners thereof with heterogeneous or homogeneous phase form.Heterogeneous assays comprises destroys the response gene product with DNA or binding partners is anchored on the solid phase, and detects the mixture that is anchored on the solid phase when reaction finishes.In homogeneous determination, in liquid phase, carry out entire reaction.In any method, can change the order that adds reactant, to obtain different information about the compound of test.For example, can identify by the following method by for example competing the interactional test compounds of disturbing DNA to destroy between response gene product and the binding partners: in the presence of test substances, react, promptly by before adding DNA destruction reactive protein and interactional binding partners or simultaneously test substances is added reaction mixture.Perhaps,, can detect the test compounds of destroying preformed mixture, as the compound with higher binding constant of one of the composition of replacing mixture by after forming mixture, test compounds being added reaction mixture.Various forms has hereinafter briefly been described.

In the heterogeneous assays system, DNA is destroyed the response gene product or interactional binding partners is anchored on the solid phase, and the direct or indirect mark material of grappling not.In practice, can utilize microtiter plate easily.Can be by non-covalent or covalent attachment, substance fixed with grappling.Can finish non-covalent adhering to simply by destroying response gene product or binding partners solution bag with DNA by solid surface and dry.Perhaps, can material be anchored to solid surface with the specificity immobilized antibody for the treatment of the fixed material.Can prepare surface and storage in advance.

In order to carry out this mensuration, be with or without the surface that under the condition of test compounds the mating partner of fixed material is exposed to the bag quilt.After reaction is finished, remove unreacted composition (for example, by washing) and remain fixed in the mixture of any formation of solid surface.Can finish the detection of the mixture that is anchored on solid surface with multiple mode.When mark in advance during loose material, the detection that is fixed on lip-deep mark shows and has formed mixture.When not in advance during the loose material of mark, can detect with indirect labelling and be anchored on lip-deep mixture, for example, adopt the specific marker antibody (antibody can carry out direct mark or indirect labelling with the anti-Ig antibody of mark subsequently) of initial loose material.According to the order of adding reacted constituent, can detect and suppress the test compounds that mixture formed or destroyed preformed mixture.

Perhaps, can in liquid phase, react,, and detect mixture from unreacted component separating reaction product existing or not existing under the condition of test compounds; For example, the alloy that forms in the specificity immobilized antibody grappling solution with one of binding constituents is with the mixture of the specific marker antibody test grappling of other mating partner.Equally, according to adding the order of reagent, can identify the test compounds that suppresses mixture formation or destroy preformed mixture to liquid phase.

In a kind of alternate embodiment of the present invention, can use homogeneous determination.In the method, preparation DNA destroys the preformed mixture of response gene albumen and interactional binding partners, wherein mark DNA destroy response gene product or its binding partners, but because mixture forms, the signal that this mark produces by cancellation (referring to, the U.S. Patent No. 4,109 of Rubenstein for example, 496, it utilizes this method to carry out immunoassay).With the competition of one of material in the preformed mixture and replace the interpolation of the test substances of this material, will cause producing the signal that is higher than background.In this way, can identify that destroying DNA destroys the interactional test substances of reactive protein/binding partners.

In a kind of specific embodiment, can prepare DNA and destroy the response gene product, fix to adopt recombinant DNA technology.For example, can adopt fusion vector, as pGEX-5X-l, make DNA destroy reaction coding region and glutathione-S-transferase (GST) gene fusion, amalgamation mode makes and keeps it in conjunction with activity in the fusion rotein that obtains.Can the interactional binding partners of purifying, and be used to adopt the conventional method of implementing in this area to produce monoclonal antibody.For example, can use radio isotope by the conventional method of implementing in this area ¹²⁵The I traget antibody.In heterogeneous assays, for example GST-DNA can be destroyed the reaction fusion rotein and be anchored to gsh-sepharose 4B.Then, can with allow to take place interact and the bonded mode test compounds exist or non-existent condition under the interactional binding partners of adding.When reaction finishes, can the unconjugated material of flush away, can monoclonal antibody adding system with mark in, and it is combined with the compound composition.Can keep and the associating radioactive amount of gsh-sepharose 4B by measuring, detection DNA destroys the interaction between response gene albumen and the interactional binding partners.Test compounds successfully suppresses interactional, and the radioactivity that will cause measuring reduces.

Perhaps, can under the condition that does not have solid gsh-sepharose 4B, GST-DNA destruction reaction fusion rotein and interactional binding partners be mixed together in the liquid.Can or add test compounds in these matter interactions afterwards.Then, this mixture can be added gsh-sepharose 4B, the unconjugated material of flush away.Equally, can detect the interactional inhibition degree of DNA destruction response gene product/binding partners with the associating radioactivity of pearl by antibody and the measurement that adds mark.

In another embodiment, can adopt the peptide fragment that destroys reactive protein and/or interactional binding partners corresponding to DNA to substitute in these two kinds of full-length proteins one or both and use these technology (is under the proteic situation at binding partners) in conjunction with the territory.Can use the method for the conventional any number implemented in this area to identify and the separation and combination site.These methods include, but not limited to the mutagenesis of gene of one of proteins encoded and screening coimmunoprecipitation measure in bonded destroy.Can select then to encode anaphragmic in the gene of second kind of material in the mixture.The proteic separately Gene Sequence Analysis of encoding will disclose corresponding to the proteic zone that participates in the interactional combination.Perhaps, can a kind of albumen be anchored to solid surface with method as described above in this section, and it is also combined with its binding partners interaction, described binding partners has been used proteolytic ferment, as trypsin treatment.After the washing, comprise that small peptide in conjunction with the mark in territory can keep and solid material associates, it can separate and identify by amino acid sequencing.Equally, in case obtained the gene of coding binding partners, can carry out the peptide fragment of through engineering approaches with expressing protein to short gene fragment, it is active to detect described segmental combination then, and purifying or synthetic.

For example, but not restriction, can be according to above description in this section, destroy the reaction fusion rotein and it is combined with the glutathione agarose pearl and DNA is destroyed gene product be anchored to solid material by preparation GST-DNA.Can use radio isotope,, and use proteolytic ferment, cut as trypsinase as the interactional binding partners of 35S mark.The GST-DNA that cleaved products can be added grappling then destroys the reaction fusion rotein, and makes in conjunction with taking place.Behind the unconjugated peptide of flush away, can represent the bond material of the mark in binding partner binds territory by the known method wash-out, purifying, and analysis of amino acid sequence.The peptide that produces evaluation like this be can synthesize, or recombinant DNA technology and suitable facilitation albumen fusion adopted.

5.4.4.2. the Growth Inhibition compound of DNA disrupting agent is regulated and/or is strengthened in screening

Can further screen the expression of adjusting DNA destruction response gene and/or interactional any reagent that DNA destroys reactive protein and its binding partners, destroy the adjusting and/or the Growth Inhibition abilities of enhancing DNA disrupting agent in cell such as antibody of reactive protein as 5.4.4.1 joint compounds identified, DNA.Any suitable propagation well known in the art or growth-inhibiting mensuration can be used for this purpose.In one embodiment, candidate agent and DNA disrupting agent are applied to the cell of clone, and definite Growth Inhibition changes.Preferably, determine that with the combination of the candidate agent of different concns and different concns DNA disrupting agent Growth Inhibition changes, so that determine to cause one or more combinations of the concentration of 50% candidate agent that suppresses and DNA disrupting agent, that is, and IC ₅₀

In a kind of preferred embodiment, with the MTT proliferation assay (referring to, van deLoosdrechet for example, et al., 1994, J.Immunol.Methods 174:311-320; Ohnoet al., 1991, J.Immunol.Methods 145:199-203; Ferrari et al., 1990, J.Immunol.Methods 131:165-172; Alley et al., 1988, Cancer Res.48:589-601; Carmichael et al., 1987, Cancer Res.47:936-942; Gerlieret al., 1986, J.Immunol.Methods 65:55-63; Mosmann, 1983, J.Immunological Methods 65:55-63) screens with the combination of DNA disrupting agent and be used for cytostatic candidate agent.Candidate agent and DNA disrupting agent with selected concentration were handled cell 4-72 hour.The 3-of cell and appropriate amount (4,5-dimethylthiazole-2-yl)-2 then, 5-phenylbenzene tetrazolium bromide (MTT) incubation 1-8 hour together makes the cell of survival that MTT is converted into insoluble first Cell in settling.After removing the excessive MTT that comprises in the supernatant liquor, add suitable MTT solvent, as DMSO solution, with the dissolving first By determining the optical density(OD) at 570nm place, measure the proportional MTT concentration of number then with survivaling cell.Can measure the candidate agent of a large amount of different concns, so that determine to cause 50% candidate agent that suppresses and the concentration of DNA disrupting agent.

In another kind of preferred embodiment, use the alamarBlue of cell proliferation ^TMMeasure screen can be used for cytostatic one or more candidate agents (referring to, Page etal. for example, 1993, Int.J.Oncol.3:473-476).AlamarBlue ^TMMeasure and measure cellular respiration, and measuring used as viable cell number.The interior environment of proliferative cell has higher reductibility than the interior environment of non-proliferative cell.For example, in breeding, NADPH/NADP, FADH/FAD, the ratio of FMNH/FMN and NADH/NAF increases.These metabolic intermediates can reduce alamarBlue, therefore, can be used to monitor cell proliferation.Cell number in the sample of the processing of measuring by alamarBlue can be expressed as the per-cent with respect to the cell number in the untreated control sample.

In a kind of particular, carry out alamarBlue ^TMMeasure, decide the transfection titre curve of siRNA that DNA destroys response gene whether owing to select the DNA disrupting agent of concentration, change as the existence of camptothecine to determine target.Decide the siRNA transfectional cell that DNA destroys response gene with target.After the siRNA transfection 4 hours, under the condition that has or do not exist the DNA disrupting agent, add the DMEM/10% foetal calf serum in 100 microlitres/hole, with plate incubation 68 hours under the condition of 37 ℃ and 5%CO2.From the hole, remove substratum, and with containing 10% (vol/vol) alamarBlue ^TMReagent (Biosource International Inc., Camarillo, CA) and the DMEM/10% foetal calf serum (Invitrogen) of the 1M Hepes damping fluid tissue culture reagent (Invitrogen) of 0.001 volume replace.With plate 37 ℃ of incubations 2 hours, adopt then Softmax Pro 3.1.2 software (MolecularDevices) SpectraMax plus read the plate device (Molecular Devices, Sunnyvale, CA) on, 570 and the 600nm wavelength readings.Decide the reduction per-cent in hole of the siRNA transfection that DNA destruction reacts and the hole of luciferase siRNA transfection compares with existing or do not exist under the condition of DNA disrupting agent with the target of certain titre.The % reduction numerical value that when not having the DNA disrupting agent hole of 0nM luciferase siRNA transfection is calculated is considered to 100%.

5.4.4.3. compounds identified

Compounds identified comprises in this screening has proved that DNA destroys the compound that the Growth Inhibition ability of DNA disrupting agent was expressed and regulated and/or strengthened to response gene in the selectivity adjusting cell.These compounds include, but not limited to siRNA, antisense molecule, ribozyme, triple helical, antibody and peptide molecule, aptamrs and little organic or inorganic molecule.

Compounds identified comprises that also regulating DNA destroys the interactional compound that reacts with other albumen or molecule in this screening.In one embodiment, compounds identified is to regulate the interactional compound that DNA destroys reactive protein and its interaction mating partner in this screening.In another embodiment, compounds identified is to regulate the interactional compound that DNA destroys response gene and transcriptional regulatory agent in this screening.

5.4.5. diagnosis

Several different methods can be used to diagnose with the prognostic assessment because DNA destroys cell that the adjusting defective of reaction causes to the DNA disrupting agent, as the Growth Inhibition resistance of camptothecine be used to identify to have the experimenter who the growth-inhibiting effect of DNA disrupting agent is had the tendency of resistance.

In one embodiment, this method comprises determines that DNA destroys the response gene expression level in the cell, and the expression level that wherein is higher than predetermined threshold level shows that cell has DNA disrupting agent resistance.Preferably, predetermined threshold level is at least 2 times, 4 times, 8 times or 10 times of the DNA normal expression level of destroying response gene.In another embodiment, the invention provides the method for the DNA disrupting agent resistance in the assessment cell, comprise the abundance level of determining to be destroyed by DNA in the cell response gene encoded protein, the abundance water-glass clear-cells that wherein is higher than predetermined threshold level has DNA disrupting agent resistance.In another embodiment, the invention provides the method for the DNA disrupting agent resistance in the assessment cell, comprise the activity level of determining to be destroyed by DNA in the mammalian cell response gene encoded protein, the activity level that wherein is higher than predetermined threshold level shows that cell has DNA disrupting agent resistance.Preferably, abundance or active predetermined threshold level are that DNA destroys the normal abundance of reactive protein or at least 2 times, 4 times, 8 times or 10 times of activity level.

Described method is passable, for example, uses reagent such as DNA destruction response gene nucleotide sequence and at the interaction gene product, comprises the antibody of its peptide fragment.Particularly, described reagent can be used for, and for example (1) is detected DNA and destroyed the existence that suddenlys change in the response gene, or the mRNA of detection DNA destruction response gene is not enough with respect to the overexpression or the expression of normal expression level; (2) detect DNA and destroy the response gene product destroys the reactive protein normal level with respect to DNA excess abundance or abundance deficiency.

For example, can comprise at least a specific DNA described herein by use destroys the test kit that response gene nucleic acid or anti-DNA destroy reaginic antibody reagent and carries out method described herein, described method can be advantageously used in for example clinical the setting, has with DNA with diagnosis and destroys relevant illness of response gene or unusual patient.

Destroy the reaction sudden change in order to detect DNA, the cell of any tool nuclear can be used as the initial source of genomic nucleic acids.In order to detect expression or the DNA destruction response gene product that DNA destroys response gene, can use any cell type or the tissue of having expressed DNA destruction response gene.

Detection technique based on nucleic acid is described in hereinafter 5.4.5.1 joint.The peptide detection technique is described in hereinafter 5.4.5.2 joint.

5.4.5.1.DNA destroy the detection that response gene is expressed

Can utilize the expression of DNA destruction response gene in many technology for detection cell or tissues, for example DNA destroys the cell levels of response gene transcript and/or the existence or the shortage of sudden change.Can will be used as the starting point of described determination techniques from the nucleic acid of any tool karyocyte, and can be according to well known to a person skilled in the art that the standard nucleic acid preparation procedure separates.For example, can use all to comprise the expression level that DNA destroys one or more polynucleotide probes measurements DNA destruction response gene of the nucleotide sequence in the response gene, determine that DNA destroys the expression level of response gene.In particularly preferred embodiment of the present invention, this method is used for the resistance of diagnosing human cancer to the treatment of employing DNA disrupting agent.

The hybridization or the amplification assay that DNA can be used for biological sample relate to structure unusual that DNA destroys response gene with detection, comprise point mutation, insertion, disappearance and chromosome rearrangement.Described mensuration can include, but not limited to Southern and analyze single-strand conformation polymorphism analysis (SSCP), dna microarray analysis and pcr analysis.

Being used to detect the described diagnostic method that DNA destroys the response gene specific mutant can comprise, for example, contact and incubation comprise the nucleic acid of recombinant DNA molecules, cloned genes or its degeneracy variant, it is available from sample, as patient's sample or other the suitable cell source derived from the nucleic acid reagent with one or more marks that comprise recombinant DNA molecules, cloned genes or its degeneracy variant, the conditions favouring of contact and incubation carries out specificity annealing with its complementary sequence in these reagent and DNA destruction response gene.Preferably, the length of these nucleic acid reagents is 15-30 Nucleotide at least.Behind the incubation, all unannealed nucleic acid are removed from nucleic acid: DNA destruction reaction molecular crossbred.Detect exist (if having any described molecule) of the nucleic acid carried out hybridization then.Adopt described detection scheme, the nucleic acid from purpose cell type or tissue can be fixed on the solid support of film for example, or be fixed on the frosting on the surface of microtiter plate for example or polystyrene bead.In this case, behind incubation, can easily the nucleic acid reagent of unannealed mark be removed.With well known to a person skilled in the art standard technique, the DNA that has finished remaining, annealed, mark destroys the detection of reaction nucleic acid reagent.Destroying response gene with nucleic acid reagent annealed DNA can compare with the annealing pattern of destroying the expectation of response gene sequence from normal DNA, so that determine whether to exist DNA to destroy the response gene sudden change.

The alternative diagnostic methods that the DNA that is used for detecting patient's sample or other suitable cell source destroys response gene specific nucleic acid molecule can comprise their amplification, for example pass through pcr amplification (at Mullis, K.B., 1987, U.S. Patent No. 4,683,202), then with the molecule that well known to a person skilled in the art the technology for detection amplification.Desired those compare under the situation that the DNA of normal copy destroys response gene if can be with the extension increasing sequence that obtains only contain during with nucleic acid amplification, so that determine whether to exist DNA to destroy the response gene sudden change.

Destroy in the nucleotide sequence of response gene in described hybridization and/or the preferred DNA of pcr analysis, comprise those that detect DNA destruction response gene splice site sudden change existence.

In addition, can carry out known gene type assay technology, carry the individuality that DNA destroys the response gene sudden change with evaluation.Described technology comprises, for example, uses restrictive fragment length polymerphism (RFLPs), and it comprises the sequence variations in one of the recognition site of specificity restriction enzyme of use.

In addition, described the method for analyzing the dna polymorphism that can be used for identification of dna destruction reaction sudden change, described method is utilized the existence of the short polyphone reiterated DNA sequences of different numbers between the restriction enzyme sites.For example Weber (U.S. Patent No. 5,075,217 is introduced the document as a reference in full at this) has described the dna marker thing based on length polymorphism in the short polyphone of (dC-dA) n-(dG-dT) n tumor-necrosis factor glycoproteins block.The equipartition of estimating (dC-dA) n-(dG-dT) n block is 30,000-60,000bp.Closely adjacent marker shows high frequency heredity altogether on the space, and particularly useful to identifying such as the transgenation of the sudden change in the DNA destruction response gene disease and the illness relevant with DNA destruction reaction sudden change with diagnosis.

Caskey etc. (U.S. Patent No. 5,364,759 is introduced the document as a reference in full at this) have described the DNA spectrum analysis that is used to detect short three and TTTC.This method comprises the extraction target DNA, destroys response gene as DNA, the DNA that amplification is extracted, and the mark tumor-necrosis factor glycoproteins is to form the genotype collection of illustrative plates of individual DNA.

Also can measure the expression level that DNA destroys response gene.For example, can separate and utilize the check of hybridization described above or round pcr known or suspect that expressible dna destroys the cell type or the tissue of response gene, as the RNA of the cancer cell type of performance DNA disrupting agent resistance.Isolated cells can be derived from cell culture or patient.The analysis of taking from the cell of culture can be the part that is used as based on the gene therapy technology of cell, or the check compound is to the steps necessary in the cell assessment of the effect of DNA destruction response gene expression.Described analysis can disclose the quantitative and qualitative aspect that DNA destroys the expression pattern of response gene, comprises that DNA destroys activation or deactivation that response gene is expressed.

In a kind of embodiment of described detection scheme, from purpose RNA molecule synthesis cDNA molecule (as, by being cDNA with RNA molecule reverse transcription).Then the sequence in the cDNA is used as nucleic acid amplification reaction, as the template of uses such as pcr amplification reaction.Be selected from DNA destruction response gene nucleic acid reagent as the reverse transcription of this method and the nucleic acid reagent of the synthetic initial reagent (as primer) in the nucleic acid amplification step.The preferred length of described nucleic acid reagent is 9-30 Nucleotide at least.In order to detect amplified production, can carry out nucleic acid amplification with radioactivity or nonradioactive labeling's Nucleotide.Perhaps, can prepare enough amplified productions, make and can pass through to use any suitable nucleic acid staining method, as, product shown by the standard ethidium bromide staining.

In addition, can " original position " carry out described DNA and destroy response gene and express and measure, that is, directly the tissue slice of the patient tissue that obtains from biopsy thing or excision thing (fixing and/refrigerated) be measured, thereby needn't be carried out nucleic acid purification.The nucleic acid that DNA can be destroyed response gene as the probe in the described original position program and/or primer (referring to, Nuovo for example, G.J., 1992, " PCR In Situ Hybridization:Protocols And Applications ", Raven Press, NY).

Perhaps,, can carry out standard Northern and analyze, destroy the mRNA expression level of response gene to determine DNA if can obtain the suitable cell of capacity.

Also can destroy response gene and express, destroy the cell levels of reaction transcript and/or the existence or the shortage of sudden change as DNA with the DNA in the dna microarray technical evaluation cell or tissue.In described technology, one or more polynucleotide probes that all comprise the sequence of DNA destruction response gene can be used for the expression that monitoring of DNA is destroyed response gene.Therefore, the invention provides the dna microarray that comprises polynucleotide probes, described probe comprises the sequence that DNA destroys response gene.

Any type of dna microarray technology can be used in combination with the present invention.In one embodiment, by DNA being destroyed the cDNA fragment of response gene, be deposited on the suitable surface as the PCR product of full-length cDNA, ESTs etc., preparation spot cDNA microarray (referring to, DeRisi et al. for example, 1996, Nature Genetics 14:457-460; Shalon etal., 1996, Genome Res.6:689-645; Schena et al., 1995, Proc.Natl.Acad.Sci.U.S.A.93:10539-11286; With Duggan et al., Nature GeneticsSupplement 21:10-14).In another embodiment, by photoetching technique from the teeth outwards original position synthetic contain with DNA destroy the sequence complementary oligonucleotide of response gene high density oligonucleotide array (referring to, for example, Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc.Natl.Acad.Sci.U.S.A.91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; McGall et al., 1996, Proc.Natl.Acad.Sci.U.S.A.93:13555-13560; United States Patent(USP) Nos. 5,578,832; 5,556,752; 5,510,270; 5,858,659; With 6,040,138).The microarray technology of this form to detect single nucleotide polymorphism (SNPs) particularly useful (referring to, Hacia et al. for example, 1999, Nat Genet.22:164-7; Wang et al., 1998, Science280:1077-82).In another embodiment, by ink-jet technology from the teeth outwards original position synthetic contain with DNA destroy the sequence complementary oligonucleotide of response gene high density oligonucleotide array (referring to, Blanchard for example, the open WO 98/41531 of disclosed international patent application on September 24th, 1998; Blanchard et al., 1996, Biosensors andBioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arraysin Genetic Engineering, Vol.20, J.K.Setlow, Ed., Plenum Press, New York at pages 111-123).In another embodiment, the dna microarray that allows to carry out the control of electronics severity can be used in combination with the polynucleotide probes of the sequence that comprises DNA destruction response gene (referring to, for example U.S. Patent No. 5,849, and 486).

5.4.5.2.DNA destroy the detection of response gene product

Can be according to description herein, the DNA of anti-wild-type or sudden change is destroyed diagnosis and the prognosis agent of the antibody of response gene product or its conservative variant or peptide fragment as DNA disrupting agent resistance.Described diagnostic method can be used to detect expression level unusual that DNA destroys response gene, or DNA destroys time, tissue, cell or Subcellular Localization unusual of the structure of response gene product and/or product.

Because it is intracellular gene product that DNA destroys the response gene product, antibody described below and method of immunity destroy the illness that the adjusting defective of response gene causes at assessment treatment DNA, have importance in the external application as the effect of proliferative disease.Antibody as mentioned below or antibody fragment can be used for the possible treatment compound of in-vitro screening, to determine them DNA are destroyed the effect that response gene is expressed and DNA destruction reaction peptide is produced.Can identify that DNA is destroyed the relevant illness of adjusting defective of reacting has the compound of beneficial effect, and determine the treatment effective dose.

Also can use for example external method of immunity to assess the effectiveness of DNA being destroyed the relevant illness of the adjusting defective of reaction based on the gene therapy of cell.The antibody that can external use anti-DNA destroys the reaction peptide is determined to destroy the DNA that finishes in the cell of reaction peptide and destroy the response gene expression level to produce DNA genetically engineered.Evidence disclosed herein shows that it is gene product in the cell that DNA destroys the response gene product, and described mensuration is preferably carried out with cell lysate or extract.Described analysis makes it possible to determine to obtain the necessary transformant number of interior curative effect, and the optimized gene replacement scheme.

Tissue to be analyzed or cell type generally include known or suspect that expressible dna destroys those of response gene, as, DNA disrupting agent resistance cancer cell type.The protein separating method of Shi Yonging can be herein, for example, those that describe among Harlow and the Lane (Harlow, E.and Lane, D., 1988, " Antibodies:A Laboratory Manual ", ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York), be incorporated herein by reference in full at this.Isolated cells can be derived from cell culture or patient.The analysis of taking from the cell of culture can be used to check compound that DNA is destroyed the effect that response gene is expressed.

Being used to detect the preferred diagnostic method that DNA destroys response gene product or its conservative variant or peptide fragment can comprise, for example immunoassay, wherein by DNA destroy response gene product or its conservative variant or peptide fragment and anti-DNA destruction response gene product specific antibody interaction partners its detect.

For example, antibody or the antibody fragment that destroys reactive protein in conjunction with DNA can be used for the existence of DNA destruction response gene product or its conservative variant or peptide fragment is carried out quantitatively or qualitative detection.This can (referring to this joint hereinafter) immunofluorescence technique be finished by for example use and opticmicroscope, flow cytometry or the fluorescently-labeled antibody of fluoroscopic examination bonded.If described DNA destroys the response gene product at cell surface expression, described technology is particularly preferred.

Be used for antibody of the present invention (or its fragment) and can additionally carry out the histology use, as be used for immunofluorescence or immunoelectron microscope, be used for the in situ detection that DNA destroys response gene product or its conservative variant or peptide fragment.Can be by removing histological specimen from the patient, and, finish in situ detection with its antibody that is applied to mark of the present invention.Preferably cover on the biological sample and administration of antibodies (or fragment) by antibody (or fragment) with mark.By using described program, can determine that not only DNA destroys the existence of response gene product or its conservative variant or peptide fragment, also can determine its distribution in being examined tissue.Employing the present invention those skilled in the art will readily recognize that, can improve in the multiple Histological method (as dyeing procedure) any one, to finish described in situ detection.

The immunoassay that DNA destroys response gene product or its conservative variant or peptide fragment are usually included in the antibody that can identification of dna destroys the detectability mark of response gene product or its conservative variant or peptide fragment and have incubation sample down, as biofluid, tissue extract, the cell of new results or the lysate of the cell that incubation is crossed in cell culture, and by any technology for detection bonded antibody well known in the art.

Can make biological sample contact and be fixed on solid support or carrier, as nitrocellulose or other can fixed cell, on the solid support of cell granulations or soluble protein.Can handle with the DNA destruction reactive protein specific antibody of detectability mark subsequently with suitable damping fluid washing upholder then.Can wash solid support to remove unconjugated antibody with damping fluid for the second time then.Can detect the amount of the bonded marker on the solid support then by ordinary method.

" solid support or carrier " is intended to represent can conjugated antigen or any upholder of antibody.Known upholder or carrier comprise glass, polystyrene, polypropylene glycol, polyoxyethylene glycol, dextran, nylon, amylase, natural and Mierocrystalline cellulose, polyacrylamide, gabbro and the magnetite modified.The character of carrier can be soluble to a certain degree or insoluble, to be used for purpose of the present invention.Support material can have any possible node configuration substantially, as long as the link coupled molecule can conjugated antigen or antibody.Therefore, the upholder configuration can be a spheric, as pearl or cylindrical, as is positioned at the outside surface of test trough internal surface or bar.Perhaps, the surface can be flat, as sheet, test strip etc.Preferred upholder comprises polystyrene bead.Those skilled in the art will know that to be used for binding antibody or antigenic many other suitable carriers, maybe can determine by using normal experiment.

Can determine that given batch anti-DNA destroys the combination activity of response gene product antibody according to known method.Those skilled in the art can determine each operating and optimum condition determination of measuring by normal experiment.

One of approach that can the detectability marker DNA destroys response gene peptide specific antibody is by it is connected with enzyme, and be used for enzyme immunoassay (EIA) (Voller, A., " TheEnzyme Linked Immunosorbent Assay (ELISA) ", 1978, DiagnosticHorizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A.et al., 1978, J.Clin.Pathol.31:507-520; Butler, J.E., 1981, Meth.Enzymol.73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL; Ishikawa, E.etal., (eds.), and 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo).With the enzyme of antibodies will with suitable substrate, preferred chromophoric substrate reaction, reactive mode makes that generation can be by spectrophotometric for example, fluorescent method or the chemical part by the detection of visual inspection method.The enzyme that can be used to detection property traget antibody comprises, but be not limited to malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, Δ-5-steroidal class isomerase, Alcohol Dehydrogenase from Yeast, alpha-phosphate glyceryl ester, desaturase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase enzymes, rnase, urease, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.The colorimetry of chromophoric substrate that can be by using enzyme is finished detection.Also can finish this detection by the enzymatic reaction degree of naked eyes comparison to the substrate of the standard substance of similar preparation.

Also can finish detection with in multiple other immunoassay any one.For example, by radiolabelled antibody or antibody fragment, can by use radioimmunoassay (RIA) detect DNA destroy the reaction peptide (referring to, Weintraub for example, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March 1986, are hereby incorporated by).Can be by such as using gamma counter or scintillometer or by autoradiographic method detection of radioactive isotropic substance.

Also can use the fluorescent chemicals traget antibody.When fluorescently-labeled antibody is exposed to the light time with suitable wavelength, then because fluorescence can detect its existence.In most of normally used fluorescently-labeled compounds, comprise fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, Phthalyldicarboxaldehyde and fluorescamine.

Also can use the fluorescent emission metal, as ¹⁵²Eu or other lanthanide series metal detectability traget antibody.Can use metal-chelating group that these metals are connected with antibody such as Diethylenetriamine valeric acid (DTPA) or ethylenediamine tetraacetic acid (EDTA) (EDTA).

Also can be by antibody and chemiluminescence compound coupling are detected antibody.Determine the existence of the antibody of chemiluminescent labeling then by the existence that detects the fluorescence that in chemical reaction process, produces.The example of useful especially chemiluminescent labeling compound is luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, heromatic acridinium ester, imidazoles, acridinium salt and barkite.

Equally, can use bioluminescent compound mark antibody of the present invention.Noclilucence is a class chemoluminescence of finding in the biosystem, and wherein catalytic protein has increased the efficient of chemiluminescence reaction.By detecting luminous existence, determine the existence of bioluminescent protein.The important biomolecule luminophor that is used for the mark purpose is fluorescein, luciferase and aequorin.

Destroy the method that response gene is expressed 5.4.6. regulate DNA

Can use and regulate the expression that DNA destroys response gene in the multiple therapy methods body according to the present invention.For example, can be to the siRNA molecular engineeringization, and be used for making in vivo DNA to destroy the response gene silence.Also can carry out through engineering approaches, and be used for the translation of blocking dna destruction reaction mRNA in the body the antisense DNA molecule.Perhaps, can design ribozyme molecule, destroy reaction mRNA with cutting in the body and destruction DNA.In another kind of scheme, design oligonucleotides is distinguished (district that comprises the encoding sequence upstream) hybridization so that destroy 5 ' of response gene with DNA, and formation can be used to block or reduce the triple-helix structure that DNA destruction response gene is transcribed.If desired, also can design oligonucleotides so that with the binding site hybridization of negative conditioning agent with form triple-helix structure, so that the combining and strengthen transcribing of DNA destruction response gene of blocking-up and negative conditioning agent.

In a kind of preferred embodiment, design siRNA, antisense molecule, ribozyme and triple helical Nucleotide to be suppressing one or more DNA and destroy the translation of reaction isoforms or to transcribe, and to other may to destroy other expression of gene influences of the total one or more motifs of response gene minimum with DNA.For reaching this purpose, should destroy the distinctive correlated series of response gene based on DNA and design employed oligonucleotide.

For example, but be not restriction, oligonucleotide should not drop on DNA and destroy the nucleotide sequence of response gene and the highest zone of nucleotide sequence homology of its gene.Under the situation of antisense molecule, described sequence preference is selected from tabulation above.The length of sequence is at least 18 Nucleotide preferably, so that the enough strong annealing of realization and said target mrna sequence, thereby the translation of prevention sequence.Izant?et?al.，1984，Cell，36：1007-1015；Rosenberg?et?al.，1985，Nature，313：703-706。

Under the situation of " tup " type ribozyme, the target sequence of ribozyme is preferably selected from tabulation above.Ribozyme is the active RNA molecule of endonuclease with high special.Hammerhead ribozyme comprises at least a portion complementary hybridization region of nucleotide sequence and target RNA and is fit to the catalytic domain of cutting target RNA.Hybridization region comprises nine (9) individual or more a plurality of Nucleotide.Therefore, hammerhead ribozyme of the present invention has and sequence complementary hybridization region listed above, and length is at least 9 Nucleotide.The structure of described ribozyme and production are well known in the art, and are described in Haseloff et al., 1988, Nature, 334:585-591 more completely.

Ribozyme of the present invention also comprises RNA endonuclease (after this being called " Cech type ribozyme "), as natural existence the in Tetrahymena Thermophila (being called IVS or L-19IVS RNA), and the ribozyme (Zaug that is fully described by Thomas Cech and co-worker thereof, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; The disclosed international patent application No.WO88/04300 of UniversityPatents Inc.; Been et al., 1986, Cell, 47:207-216).The Cech endonuclease has 8 base pair avtive spots with target RNA sequence hybridization, and the cutting of target RNA is taking place thereafter.

For with DNA destroy response gene 5 ' terminal hybridization and with its formation triple-helix structure, and can be used to block the oligonucleotide of transcribing, they preferably destroy the sequence complementation of 5 ' end of non-existent DNA destruction response gene in the reacting phase correlation gene with impregnable other DNA of expression level.Described sequence preferably do not comprise yet DNA destroy in the reaction promotor in addition slightly with the zone of described other dna homolog.Can use aforesaid compound by several different methods well known in the art, include, but not limited to use liposome as delivery vectors.When being in anti-degraded form, also can use naked DNA or RNA molecule, described anti-degraded is by modifying end, by forming ring molecule, or by using alternating bond, comprises the key that phosphorothioate bond and thiophosphoryl are modified.In addition, can pass nucleic acid, wherein nucleic acid molecule be puted together in polylysine or Transferrins,iron complexes by promoting transhipment to send.Also can pass through multiple virus vector, include, but are not limited in retrovirus, vaccinia virus, AAV and the adenovirus any one, with nucleic acid delivery in cell.

Perhaps, can make up the recombinant nucleic acid molecules of encoding or destroying the response gene nucleic acid molecule as described antisense molecule, ribozyme, triple helical or DNA.This nucleic acid molecule can be RNA or DNA.If nucleic acid encoding RNA, this sequence preference is connected with the regulatory element operability, so that produce enough RNA products of the needs of copy.Regulatory element can allow the composing type of sequence or adjustment type to transcribe.Can be in vivo, that is, the transfer vector of one or more RNA that in the cell of biology, will encode, as bacterial plasmid or viral RNA or DNA transfection in cell.(as, Llewellyn et al., 1987, J.Mol.Biol., 195:115-123; Hanahanet al.1983, J.Mol.Biol., 166:557-580).In case be positioned at cell, transfer vector can duplicate, and is transcribed by the cell aggregation enzyme, to produce RNA, perhaps can be integrated into the genome of host cell.Perhaps, can pass through the micromanipulation technology, as micro-injection, the transfer vector transfection of sequence that will comprise one or more RNA that encode makes transfer vector or its be partially integrated in the genome of host cell in cell or transfered cell.

RNAi also can be used for the expression that knockout dna destroys response gene.In one embodiment, be used to the mRNA that degrades with the double stranded rna molecule of 21-23 Nucleotide of the homologous region hybridization that destroys the mRNA that response gene transcribes from DNA, thereby make the expression " silence " of DNA destruction response gene.Preferably, dsRNA has hybridization region, as, the double stranded region of 19 Nucleotide, itself and DNA destroy the sequence complementation of the encoding sequence of response gene.Any target can be decided DNA and destroy response gene, be used for the present invention as the siRNA of the suitable encoding sequence of human STK6 or TPX2 gene.As exemplary embodiment, according to the Standard Selection rule (referring to, Elbashir et al. for example, 2002, Methods 26:199-213 is incorporated herein by reference in full at this) the design target decide the double-stranded siRNA of 21 Nucleotide of the coding region of DNA destruction response gene.

Can use any standard method that is used to import siRNA.In one embodiment, the induced gene silence by the siRNA that decides DNA destruction response gene to the presented by cells target (referring to, for example, Elbashir et al., 2001, Nature 411,494-498; Elbashir et al., 2001, Genes Dev.15,188-200 introduces all above-mentioned documents as a reference in full at this).SiRNA can chemosynthesis, perhaps derived from by the cutting of reorganization nickase to double-stranded RNA.Being used to import the another kind of method that makes DNA destroy the double-stranded DNA (dsRNA) of response gene silence is shRNA, that is, short hairpin RNA (referring to, Paddison et al. for example, 2002, Genes Dev.16,948-958; Brummelkamp et al., 2002, Science296,550-553; Sui, G.et al.2002, Proc.Natl.Acad.Sci.USA 99,5515-5520 introduces all above-mentioned documents as a reference in full at this).In the method, express target from plasmid (or virus) and decide the siRNA that DNA destroys response gene, it is as inverted repeats, has the ring sequence that interleaves with the formation hairpin structure.The rna transcription thing that contains hair clip that obtains by nickase processing is used for reticent siRNA with generation subsequently.Can be in cell stably express based on the shRNA of plasmid, make and can in cell, carry out long-term gene silencing in vitro and in vivo (referring to, McCaffrey et al.2002, Nature 418,38-39; Xia et al., 2002, Nat.Biotech.20,1006-1010; Lewis et al., 2002, Nat.Genetics 32,107-108; Rubinson et al., 2003, Nat.Genetics 33,401-406; Tiscornia et al., 2003, Proc.AM.Acad.Sci.USA100,1844-1848 introduces all above-mentioned documents as a reference in full at this).Also target can be decided to send in the siRNA body that DNA destroys response gene to be delivered to Mammals, in the organ or tissue as the mankind (referring to, Song et al.2003 for example, Nat.Medicine 9,347-351; Sorensen etal., 2003, J.Mol.Biol.327,761-766; Lewis et al., 2002, Nat.Genetics32,107-108 introduces all above-mentioned documents as a reference in full at this).In the method, with the solution intravenous injection of siRNA in Mammals.SiRNA can arrive purpose organ or tissue then, and effectively reduces target gene expression in mammalian organs or the tissue.

5.4.7. regulate the method that DNA destroys active and/or its approach of reactive protein

Can destroy the activity that DNA destruction reactive protein is regulated in the interaction between reactive protein and its binding partners by regulating DNA.In one embodiment, can use reagent, as the combination of binding partners as described in antibody, aptamers, the little organic or inorganic molecules in inhibiting, so that regulate DNA disrupting agent resistance.In another embodiment, can use reagent, destroy proteic activity in the reactive protein adjusting approach as antibody, aptamers, little organic or inorganic molecules in inhibiting DNA, so that regulate DNA disrupting agent resistance.In one embodiment, use kinase inhibitor, regulate DNA as Herbimycin, Gleevec, Genistein, Lavendustin, Iressa and destroy the kinase whose activity of reactive protein.

5.4.8. decide by target that DNA destroys response gene and/or its product carries out cancer therapy

Can destroy that reaction is expressed and/or active method and/or composition and the combination therapy of DNA disrupting agent suffer from the patient of cancer with adjusting mentioned above DNA.Particularly, described method and/or composition and DNA disrupting agent can be united use, be used for the treatment of to suffer from and show the patient of cancer that DNA destroys the DNA disrupting agent resistance of reaction mediation.Described therapy can be used for the treatment of cancer, includes, but not limited to rhabdosarcoma, neuroblastoma and glioblastoma, small cell lung cancer, osteosarcoma, carcinoma of the pancreas, mammary gland and prostate cancer, mouse melanoma and leukemia, and B cell lymphoma.

In preferred embodiments, method of the present invention and/or composition and DNA disrupting agent are united the patient who is used for the treatment of the cancer of suffering from the DNA disrupting agent resistance that shows DNA destruction reaction mediation.In described embodiment, regulate that DNA destroys that reaction is expressed and/or active, giving the susceptibility of cancer cells, thereby give or strengthen the validity of DNA disrupting agent treatment to the DNA disrupting agent.

In combination therapy, can be before using the DNA disrupting agent, simultaneously or use one or more compositions of the present invention afterwards.In one embodiment, before using the DNA disrupting agent, use composition of the present invention.Can determine to use timed interval between composition of the present invention and the DNA disrupting agent by the normal experiment that those skilled in the art are familiar with.In one embodiment, after reaching the threshold value that needs, DNA destruction reactive protein level gives DNA disrupting agent.Can determine that DNA destroys the level of reactive protein by adopting above-described any technology.

In another embodiment, use composition of the present invention simultaneously with the DNA disrupting agent.

In another embodiment, also after using the DNA disrupting agent, use one or more compositions of the present invention.When one or more compositions of the present invention that the long half time of DNA disrupting agent uses in treatment, described using can be useful especially.

It will be understood by those skilled in the art that the arbitrary combination of the different administration time that to use composition of the present invention and DNA disrupting agent.For example, when the long half time of DNA disrupting agent during, preferably before or after using the DNA disrupting agent, use composition of the present invention in composition of the present invention.

Use the frequency of composition of the present invention or depend on that at interval the DNA of needs destroys reaction level, can determine this level by above-described any technology.Change into the level that is higher or lower than needs when DNA destroys the reactive protein level, can increase or reduce the frequency of administration of composition of the present invention.

Can by any method assessment well known in the art separately or with the effect or the benefit of the co-administered composition of the present invention of DNA disrupting agent, for example, by requiring based on the dosage of measuring survival rate, side effect, DNA disrupting agent or the method for its arbitrary combination.If being applied in of composition of the present invention realized any one or multiple benefit among the patient, as increase survival rate, reduce side effect, reduce the dosage requirement of DNA disrupting agent, think that then composition of the present invention strengthened DNA disrupting agent treatment, and think that this method is effective.

5.5. pharmaceutical preparation and route of administration

Can use to the patient with the treatment effective dose and determine to influence STK6 genetic expression or the active compound of gene product, regulate the relevant illness of defective with STK6 with treatment or improvement.The treatment effective dose is meant is enough to cause KSPi resistance in the cell to improve and/or strengthen the amount of the Growth Inhibition compound of KSP inhibitor.

5.5.1. effective dose

Can in cell culture or laboratory animal, determine the toxicity and the curative effect of described compound by standard pharmaceutical procedures, as determining LD ₅₀(to 50% lethal dosage of colony) and ED ₅₀(colony 50% in the treatment effective dosage).The dosage ratio of toxicity and curative effect is a therapeutic index, and it is expressed as ratio LD ₅₀/ ED ₅₀The compound that the preferred therapeutic index is big.Although can use the compound that shows toxic side effects, must careful design with the send delivery system of described compound target due to the site of affected tissue so that may be minimum, thereby reduce side effect to unaffected cells injury.

Can be used to customize the dosage that is used for human certain limit from the data that cell cultures is measured and zooscopy obtains.The dosage of described compound preferably drops on and comprises having seldom or do not have toxic ED ₅₀The circulation composition scope.Dosage can change in this scope, depends on the formulation of use and the route of administration of employing.For any compound that is used for the inventive method, can measure from cell cultures at first and estimate the treatment effective dose.Can in animal model, customize dosage, to realize being included in the circulating plasma concentration range of the IC50 (that is, realizing the test compounds concentration of maximum half that suppresses of symptom) that determines in the cell culture.Described information can be used for useful dosage among more accurate definite mankind.For example, can be by the level in the high-efficient liquid phase color spectrometry blood plasma.

5.5.2. preparation and purposes

Can carry out preparationization with one or more physiology acceptable carriers or excipient to pharmaceutical composition used according to the invention according to conventional methods.

Therefore, can carry out preparationization, use or by oral, cheek or rectal administration for use in sucking or be blown into (through port or nose) to compound and the acceptable salt of physiology thereof and solvate.

For Orally administered, the form that pharmaceutical composition can adopt for example is, by ordinary method, with the acceptable excipient of pharmacy, as tackiness agent (as W-Gum, polyvinylpyrrolidone or the Vltra tears of pre-gelledization); Weighting agent (as lactose, Microcrystalline Cellulose or secondary calcium phosphate); Lubricant (as Magnesium Stearate, talcum or silicon-dioxide); Disintegrating agent (as yam starch or Vivastar P 5000); Or the tablet or the capsule of wetting agent (as sodium lauryl sulphate) preparation.Can be with method peridium patch well known in the art agent.Being used for Orally administered liquid preparation can be following form, for example, solution, syrup or suspension, or they can be used as the dryed product existence, water or other suitable excipient preparation before use.Can pass through ordinary method, adopt the pharmacy acceptable additive to prepare described liquid preparation, described additive such as suspension agent (as sorbitol syrup, derivatived cellulose or hydrogenation edible fat); Emulsifying agent (as Yelkin TTS or Sudan Gum-arabic); Non-water excipient (as the vegetables oil of Prunus amygdalus oil, grease, ethanol or fractional separation); And sanitas (as methyl p-hydroxybenzoate or propyl ester or Sorbic Acid).Described preparation also can contain suitable buffering salt, seasonings, tinting material and sweeting agent.

Be used for suitably preparationization of Orally administered preparation, to obtain the controlled release of active compound.

For using through cheek, composition can be taked the tablet or the lozenge form of preparationization in a conventional manner.

Use for suction, compound used according to the invention is routinely with the aerosol spray form, send by pressurized package or atomizer and to pass, wherein use suitable propelling agent, as Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas.Under the situation of pressurized aerosol, can determine dose unit by valve is provided, to send the amount of passing metering.Can prepare and contain compound and suitable powder base, as the capsule and the cartridge case of for example gelatin that is used for sucker or insufflator of the powdered mixture of lactose or starch.

Can carry out preparationization to compound, be used for, for example pass through bolus injection or continuous infusion and parenteral administration by injection.The preparation that is used to inject may reside in unit dosage, for example, in ampoule or multi-agent container, wherein adds sanitas.Composition can be such as the suspension in oiliness or the aqueous carrier, solution or form of emulsion, and can contain preparation reagent, as suspension agent, stablizer and/or dispersion agent.Perhaps, activeconstituents can be a powder type, is used for using before use suitable carriers, as aseptic pyrogen-free water preparation.

Also can be in rectal compositions, as in suppository or the enema,retention compound carry out preparationization, described suppository or enema,retention for example can contain conventional suppository base, as cocoa ester or other glyceryl ester.

Except previously described preparation, also compound formulation can be turned to the storage preparation.Described prolonged action preparation can be by implanting (for example subcutaneous or intramuscular) or using by intramuscularly.Therefore, for example, can be with suitable polymerization or hydrophobic material (for example as the milk sap that can accept in the oil) or ion exchange resin, or as the slightly soluble derivative, for example as slightly soluble salt pair compound formulationization.

If desired, described composition may reside in packing or the distribution device, and described device can comprise the one or more unit dosage that contain activeconstituents.Packing can for example comprise metal or plastic foil, for example blister package.Can be in packing or the distribution device with using explanation.

5.5.3. route of administration

Suitable route of administration can comprise, for example oral, rectum, through mucous membrane, use through skin or enteron aisle; Stomach is sent outside and is passed, and comprises intramuscular, subcutaneous, intramedullary injection, and in the sheath, directly in the ventricle, in the intravenously, intraperitoneal, nose or intraocular injection.

Perhaps, can use, for example, compound is injected directly into affected area, usually to store or extended release preparation at the topical application compound rather than with system mode.

In addition, can be in the fixed drug delivery system of target, for example, at bag by drug administration in the liposome of specific antibody of influenced cell.The liposome target is scheduled cell, and absorbed by cell selective.

5.5.4. packing

If desired, composition may reside in the packing or distribution device that can comprise the one or more unit dosage that contain activeconstituents.Packing can for example comprise metal or plastic foil, for example blister package.Can be in packing or the distribution device with using explanation.Also can prepare and comprise the composition that comprise The compounds of this invention of preparationization in compatible pharmaceutical carriers, be placed in the suitable containers, and stick the label that is used for the treatment of the indication of pointing out.The appropriate conditions of pointing out on the label can comprise the treatment disease, as be characterised in that unusual or excessive STK6 or DNA destroy that response gene is expressed or active those.

6. embodiment

Following examples are for the present invention being described, being not intended to limit by any way the present invention.

6.1. embodiment 1:STK6 and TPX2 and KSP interact

This embodiment has illustrated in the screening siRNA library and the interactional gene of KSP gene inhibition agent.CIN8 is the Saccharomyces cerevisiae homologue of KSP.The deletion mutant of CIN8 can be survived, and has identified (CIN8 exists next really not so) many genes necessary under the non-existent condition of CIN8 (Geiser et al., 1997, Mol Biol Cell.8:1035-1050).Similarly, the destruction of inferring human homology's thing of these genes may not have more destructiveness to growth of tumour cell under the existence condition than it in the presence of the KSPi of suboptimal concentrations.Screening contains adjusting KSP inhibitor (1S)-the 1-{[(2S)-4-(2 in the siRNA library that it is reported with the siRNA of the homologue of CIN8 collaborative lethal 11 kinds of gene: CDC20, ROCK2, TTK, FZR1, BUB1, BUB3, BUB1B, MAD1L1, MAD2L1, DNCH1 and STK6, the 5-difluorophenyl)-and 2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine (EC ₅₀The gene of effect about 80nM).The sequence of the siRNA of fixed these the 11 kinds of genes of target is listed in Table I.Exist or do not exist＜(1S)-1-{[(2S)-4-(2, the 5-the difluorophenyl)-2-phenyl-2 of EC10 concentration (25nM), 5-dihydro-1H-pyrroles-1-yl] carbonyl-condition of 2-methyl propylamine under with these siRNA transfections in the HeLa cell.Table I has also been listed the sequence that the difference target is decided the siRNA of luciferase, PTEN and KSP.

Be performed as follows the siRNA transfection: in transfection the day before yesterday, the cell that 100 microlitres are selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) grow to the about 90% cervical cancer HeLa cell (ATCC that converges in, Cat.No.CCL-2) with 1500 cells/well be planted in the 96 hole tissue culturing plates (Corning, Coming, NY).For each transfection, (Dharmacon Denver) mixes with siRNA from 5 microlitre serial dilutions of 20 micromole's liquid storages with 85 microlitre OptiMEM (Invitrogen).For each transfection, 5 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 10 microlitre OptiMEM/Oligofectamine mixtures and OptiMEM/siRNA mixture are assigned in each test tube, mix, and at room temperature incubation 15-20 minute.10 microlitre transfection mixtures are distributed in each hole of 96 orifice plates, and under the condition of 37C and 5%CO2 incubation 4 hours.

After 4 hours, what add 100 microlitres/hole contains or does not contain 25nM (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-and 2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-the DMEM/10% foetal calf serum of 2-methyl propylamine, with plate incubation 68 hours again under the condition of 37 ℃ and 5%CO2.Measure cell growth (referring to 5.2 joints) with Alamar Blue assay method.AlamarBlue measures and measures cellular respiration, and with observed value measuring as viable cell number.The interior environment of proliferative cell has higher reductibility than the interior environment of non-proliferative cell.Particularly, in breeding, NADPH/NADP, FADH/FAD, the ratio of FMNH/FMN and NADH/NAF increases.These metabolic intermediates can reduce alamarBlue, therefore, can be used to monitor cell proliferation.In the present embodiment, measure according to hereinafter carrying out alamarBlue, to determine 25nM KSP inhibitor (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-whether the existence of 2-methyl propylamine changed STK6siRNA transfection titration curve: after the transfection 72 hours, from the hole, remove substratum, and with containing 10% (vol/vol) alamarBlue reagent (Biosource InternationalInc., Camarillo, CA) and the DMEM/10% foetal calf serum (Invitrogen) of the 1M Hepes damping fluid tissue culture reagent (Invitrogen) of 0.001 volume replace.With plate 37 ℃ of incubations 2 hours, adopt then Softmax Pro 3.1.2 software (MolecularDevices) SpectraMax plus read the plate device (Molecular Devices, Sunnyvale, CA) on, 570 and the 600nm wavelength readings.The % reduction that contains the hole of sample is determined according to formula 1.Deduct the not % reduction in celliferous hole from the % reduction in the hole of containing sample, to determine to be higher than the % reduction of background.To there be or do not exist 25nM (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-and 2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-compare with the reduction per-cent in the hole of the STK6siRNA transfection of certain titre and the hole of the siRNA transfection that target is decided luciferase under the condition of 2-methyl propylamine.Do not have (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-the % reduction numerical value that the hole of 0nM luciferase siRNA transfection calculated during 2-methyl propylamine is considered to 100%.

At (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-existence of 2-methyl propylamine is following, and three kinds of siRNA that target is decided STK6 (STK6-1, STK6-2 and STK6-3) show the inhibition growth of tumour cell.In the three, STK6-1 shows the strongest growth inhibitory activity in initial screening.In order to study this growth inhibitory activity is because hitting or missing the target activity of siRNA uses target decide three kinds of extra siRNA of STK6, and assesses all 6 kinds of siRNA and induce STK6 silence and growth inhibiting ability.Between reticent level of STK6 and growth-inhibiting, there is good dependency (Fig. 1).This dependency shows that growth-inhibiting is because the activity that hits (that is, STK6 destroys).Then at (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl-2-methyl propylamine exist or non-existent condition under, the contrast siRNA (negative control) of titration STK6-1 and luciferase is (Fig. 2).The interpolation of KSPi has been moved to the left about 5-10 doubly with the STK6-1 dose response curve.This concentration of KSPi does not have to strengthen the effect of the cell growth that is caused by luciferase siRNA.On the contrary, the dose response curve of target being decided the siRNA of PTEN is not moved by KSPi, and described PTEN has similar effect to the STK-6 cell growth.The siRNA of other STK6 also strengthens the effect of KSPi cell growth.Thus, the destruction of KSP has strengthened the effect of STK6siRNA cell growth.The research of the combination of the siRNA by adopting STK6 and KSP has obtained the further support to this, and described combination table reveals than arbitrary independent stronger growth inhibitory activity of siRNA.Do not influence cell growth owing to be used for KSPi concentration self of these experiments, active effect shows as collaborative, rather than synergetic KSPi to STK6siRNA.

Interaction consistent with the interactional evidence of the physiology of these genes in Africa xenopus (Giet et al., 1999, J Biol Chem.274:15005-5013) between human STK6 and the KSP.Particularly, the Africa xenopus homologue co of STK6 and KSP is at the mitotic spindle utmost point, and by immunoprecipitation, albumen shows molecular association.In addition, KSP is the substrate of STK6.

The growth-inhibiting of STK6siRNA shows that this gene pairs growth of tumour cell is necessary, and supports that STK6 is the research of anti-tumor target.Show that the collaborative interactional data that cause death between STK6 inhibitor and the KSPi show, carry out combination therapy with these compounds, may be than treating more effective with any independent compound.STK6 is overexpression in human tumor usually, comprise prognosis mala mammary cancer (van ' t Veer et al., 2002, Nature.2002415:530-536).The amplification of STK6 relevant with resistance (Anand et al., 2003, Cancer Cell.3:51-62) to taxol.Because KSPi and the identical target (mitotic spindle) of taxol influence, the overexpression of STK6 can reduce the validity of KSPi equally.This possibility is consistent with results of interaction between the inhibitor that shows KSPi and STK6, and should study in the clinical development process of KSPi.For example, KSPi may not have best effect in the patient with breast cancer of prognosis mala, because these tumours are tended to overexpression STK6.

Figure 17 shows the The selection result to the gene of KSPi sensitivity.The result shows that TPX2 also interacts with KSP.Be used to make the siRNA sequence of TPX2 silence also to list in Table I.

The tabulation of Table I siRNA

STK6-1	GCACAAAAGCUUGUCUCCATT(SEQ?ID?NO：1)
		STK6-2	UUGCAGAUUUUGGGUGGUCTT(SEQ?ID?NO：2)
STK6-3	ACAGUCUUAGGAAUCGUGCTT(SEQ?ID?NO：3)
		STK6-4	CCUCCCUAUUCAGAAAGCUTT(SEQ?ID?NO：4)
STK6-5	GACUUUGAAAUUGGUCGCCTT(SEQ?ID?NO：5)
		STK6-6	CACCCAAAAGAGCAAGCAGTT(SEQ?ID?NO：6)
ROCK2-1	AACCAGUCUAUUAGACGGCTT(SEQ?ID?NO：7)
		ROCK2-2	GUGACUCUCCAUCUUGUAGTT(SEQ?ID?NO：8)
ROCK2-3	GUGGCCUCAAAGGCACUUATT(SEQ?ID?NO：9)
		CDC20-1	CCCAUCACCUCAGUUGUUUTT(SEQ?ID?NO：10)
CDC20-2	GACCUGCCGUUACAUUCCUTT(SEQ?ID?NO：11)
		CDC20-3	GGAGAACCAGUCUGAAAACTT(SEQ?ID?NO：12)
TTK-1	AUGCUGGAAAUUGCCCUGCTT(SEQ?ID?NO：13)
		TTK-2	ACAACCCAGAGGACUGGUUTT(SEQ?ID?NO：14)
TTK-3	UAUGUUCUGGGCCAACUUGTT(SEQ?ID?NO：15)
		FZR1-1	CCAGAUCCUUGUCUGGAAGTT(SEQ?ID?NO：16)
FZR1-2	CGACAACAAGCUGCUGGUCTT(SEQ?ID?NO：17)
		FZR1-3	GAAGCUGUCCAUGUUGGAGTT(SEQ?ID?NO：18)
BUB1-1	CUGUAUGGGGUAUUCGCUGTT(SEQ?ID?NO：19)
		BUB1-2	ACCCAUUUGCCAGCUCAAGTT(SEQ?ID?NO：20)
BUB1-3	CAGACUCCAUGUUUGCAGUTT(SEQ?ID?NO：21)
		BUB3-1	UACAUUUGCCACAGGUGGUTT(SEQ?ID?NO：22)
BUB3-2	CAAUUCGUACUCCCCAAUGTT(SEQ?ID?NO：23)
		BUB3-3	AGCUGCUUCAGACUGCUUCTT(SEQ?ID?NO：24)
MAD1L1-1	GACCUUUCCAGAUUCGUGGTT(SEQ?ID?NO：25)
		MAD1L1-2	AGAGCAGAGCAGAUCCGUUTT(SEQ?ID?NO：26)
MAD1L1-3	CCAGCGGCUCAAGGAGGUUTT(SEQ?ID?NO：27)
		MAD2L2-1	CCAUGACGUCGGACAUUUUTT(SEQ?ID?NO：28)
MAD2L2-2	GUGCUCUUAUCGCCUCUGUTT(SEQ?ID?NO：29)
		MAD2L2-3	ACGCAAGAAGUACAACGUGTT(SEQ?ID?NO：30)
DNCH1-1	GCAAGUUGAGCUCUACCGCTT(SEQ?ID?NO：31)
		DNCH1-2	UGGCCAGCGCUUACUGGAATT(SEQ?ID?NO：32)
DNCH1-3	GGCCAAGGAGGCGCUGGAATT(SEQ?ID?NO：33)
		BUB1B-1	AUGACCCUCUGGAUGUUUGTT(SEQ?ID?NO：34)
BUB1B-2	UGCCAAUGAUGAGGCCACATT(SEQ?ID?NO：35)
		BUB1B-3	GAAAGAACAGGUGAUCAGCTT(SEQ?ID?NO：36)
Luciferase	CGUACGCGGAAUACUUCGATT(SEQ?ID?NO：37)
		KSP-1	CUGGAUCGUAAGAAGGCAGTT(SEQ?ID?NO：38)
KSP-2	GGACAACUGCAGCUACUCUTT(SEQ?ID?NO：39)
		PTEN-1	UGGAGGGGAAUGCUCAGAATT(SEQ?ID?NO：40)
PTEN-2	UAAAGAUGGCACUUUCCCGTT(SEQ?ID?NO：41)
		PTEN-3	AAGGCAGCUAAAGGAAGUGTT(SEQ?ID?NO：42)
TPX2	UACUUGAAGGUGGGCCCAUTT(SEQ?ID?NO：1237)
		TPX2	GAAAUCAGUUGCUGAGGGCTT(SEQ?ID?NO：1238)
TPX2	ACCUAGGACCGUCUUGCUUTT(SEQ?ID?NO：1239)

6.2 embodiment 2: work in coordination with the screening that causes death with shRNA and siRNA

The present embodiment explanation makes CHEK1 and TP53 carry out the silence of RNAi mediation simultaneously, has caused the collaborative lethal effect among the human tumor cell.

Two problems have limited the potential of working in coordination with the screening that causes death with the RNAi method.At first, the proof of collaborative lethal effect need be hit and observe definite gene disruption inductive phenotype that causes death in the cell of genetically deficient or sudden change tending to first, does not then observe in only containing wild-type allele or proteic cell.Therefore, for the highly experiment of contrast, need be with hitting the clone of isogenic coupling the gene disruption to measuring collaborative lethal effect except first.For most of obtainable tumor cell lines, the clone of described coupling is to obtaining.Secondly, be subjected to the target obstruction of the siRNA of the different mRNA observations of competing each other calmly, effectively reduced the effectiveness of one or both siRNA of use by the trial of using dual siRNA transfection in cell, to produce two gene disruptions.Show in the present embodiment, can hit and send the excess revolutions of passing to dye in the liptinite of shRNA of gene, realize dual RNAi screening by use destroying first with the siRNA of fixed second gene of target.This method provides coupling (isogenic) clone to (shRNA adds deduct), and does not cause the competition between shRNA and the siRNA.In the present embodiment, set up cloned cell line, wherein the primary gene target is by the stably express silence of short hairpin RNA (shRNA).With the siRNA of fixed other gene of target these clones' transient transfection (excess revolutions is dyed) is not had obviously to influence the primary target silence that shRNA causes, shRNA does not influence the target silence that siRNA causes yet.Proved that with this method there is the collaborative lethal effect between following TP53 (p53) and the checkpoint kinase CHEK1 in lower concentration DNA disrupting agent Zorubicin.

Can by transient transfection send pass synthetic double-chain small disturbance RNA (siRNA) or from instantaneous importing or stable integration to genome recombinant vectors at cell inner expression short hairpin RNA (shRNA), thereby realization RNA interference (referring to, Paddison et al. for example, 2002, Genes Dev 16:948-958; Sui et al., 2002, Proc Natl Acad Sci USA 99:5515-5520; Yu et al., 2002, Proc Natl Acad Sci U S A 99:6047-6052; Miyagishi et al., 2002, Nat Biotechnol 20:497-500; Paul et al., 2002, Nat Biotechnol 20:505-508; Kwak et al., 2003, J Phannacol Sci 93:214-217; Brummelkamp et al., 2002, Science 296:550-553; Bodenetal., 2003, Nucleic Acids Res 31:5033-5038; Kawasaki et al., 2003, Nucleic Acids Res 31:700-707).Use coding tetracycline resistance markers and driving pRETRO-SUPER (pRS) carrier from H1 (RNA Pol III) promoter expression shRNA.The pRS-TP531026shRNA plasmid is from NKI library plasmid set deconvolution, and this is by gathering transform bacteria with this, and seeks the clone who only contains interested plasmid.Employed sequence is as follows: pRS-p53 1,026 19 aggressiveness sequence: GACTCCAGTGGTAATCTAC (SEQ ID NO:43); The primer of sequence-specific PCR: forward: GTAGATTACCACTGGAGTC (SEQ ID NO:44), oppositely: CCCTTGAACCTCCTCGTTCGACC (SEQ ID NO:45).Identify plasmid by sequence-specific PCR, and confirm by order-checking.By with FuGENE 6 (Roche) the transfection HCT116 cell that has pRS-TP53 1026 plasmids, prepare stable p 53 clones.After 48 hours cell is assigned in the 10cm culture dish that is added with the 1ug/ml tetracycline, keeps up to colony obvious (5-7 days).The clone is selected 96 orifice plates, in the 1ug/ml tetracycline, keep, and use TP53 and hGUS Pre-Developed to measure reagent (AppliedBiosystems), knock out by the TaqMan check.In order to measure instantaneous knocking out,, after 24 hours, gather in the crops RNA with Lipofectamine 2000 (Invitrogen) transfection HCT116 cell by pRS-TP53 1026 plasmids.By TaqMan assessment TP53 level.

Derive from colon tumor cell and be a plurality of tetracycline resistance TP53shRNA clones' (pRS-p53) of HCT116 the target silence (50%-96%) of analysis revealed different levels.Fig. 3 shows the TP53 expression level among clone A5 and the A11, and described clone shows the silence of highest level.TP53 silence among these clones has surpassed by transient transfection pRS-p53 to be sent and has been delivered to 24 hours observed silences (Fig. 3) behind the HCT116 cell.Transfection efficiency may the validity of restricted T P53shRNA in transient transfection.Perhaps, the cell with the reticent level of higher TP53 has obtained growth vigor in the clonal growth process.Adopt target to decide the shRNA (pRS-STK6:pRS-STK6 2,178 19 aggressiveness sequence: CATTGGAGTCATAGCATGT (SEQ ID NO:46)) of STK6, also observed the silence of the certain limit in the stable clone.But, these clones do not reach with TP53 clone in the silence of observed the same high level, its silence also is no more than the silence that obtains in instantaneous measurement.This may represent the selection of anti-high-level STK6 silence, because STK6 is the necessary gene of the growth of tumour cell in the culture.

In order to check the TP53 silence among the HCT116 clone whether to compete, dye this clone's cell with the set excess revolutions of CHEK1 specific siRNA with siRNA.The CHK1 set contains following three kinds of siRNA:CUGAAGAAGCAGUCGCAGUTT (SEQ ID NO:99); AUCGAUUCUGCUCCUCUAGTT (SEQID NO:98); And UGCCUGAAAGAGACUUGUGTT (SEQ ID NO:100).Find the reticent activity of the siRNA that the competitive TP53 of reduction of this siRNA set target is fixed.Show siRNA with 10nM or 100nM Oligofectamine (Invitrogen) transfection.For the CHK1 set,, send altogether and pass 100nM with three kinds of siRNA of 33.3nM transfection simultaneously.24 hours results RNA after transfection, and use CHK1 or TP53 and hGUS Pre-Developed to measure reagent (AppliedBiosystems), knock out by the TaqMan analysis and evaluation.Shown in Fig. 4 A, shRNA and siRNA gather the not silence of every kind of other target of competitive inhibition.Measure the inhibition that the known competitive siRNA by the shRNA of the siRNA of the transient transfection of identical sequence or stably express causes then.Shown in Fig. 4 B, three kinds of independent targets are decided KNSL1 (KNSLI210:GACCUGUGCCUUUUAGAGATT (SEQ ID NO:47); KNSLI211:GACUUCAUUGACAGUGGCCTT (SEQ ID NO:48); KNSLI212:AAAGGACAACUGCAGCUACTT (SEQ ID N0:49)) siRNA competitive inhibition is decided the silence (post on the left side) that the siRNA of the cotransfection of STK6 reaches by target.On the contrary, the influence that the silence that homology STK6shRNA causes in the clone of stable transfection is not dyed by the excess revolutions of KNSL1siRNA is even when adding competitor siRNA with high 10 times concentration also be so the post of (middle and the right).These experiments show, almost not competition between the siRNA of the shRNA of stably express and transient transfection.Observations during two kinds of different siRNA of that this competes each other with transfection together, fixed different mRNA of target is opposite, wherein effectively reduces the efficient of one or both siRNA of use.With the siRNA set transient transfection pRS and the pRS-p53HCT116 cell (embodiment 3 vide infra) of about 800 kinds of genes, and the effect of measuring cell growth by Alamar Blue assay method.Observed reaction much at one, do not shown reticent competitive inhibition the set of about 800 kinds of siRNA described in pRS cell and the pRS-p53 cell.

Subsequently, the excess revolutions that assessment CHEK1siRNA is integrated in the cell of stably express TP53shRNA is dyed, to determine whether to use it for the genetic interaction (SL) between these molecules of research.Inferred this interaction in the past, but, hindered its definite proof because shortage has enough specific reagent or gene knockout to the deduction effect of missing the target.By select to contain the clone that the A549 lung cancer cell line that free pRS carrier or pRS-p53 are arranged prepares coupling+/-TP53 expresses.A kind of cell in back shows＞90% TP53mRNA silence.Then with contrast luciferase siRNA (luc, 100nM) or CHEK1siRNA set (100nM altogether; Each 33nM of 3 kinds of siRNA) two kinds of clones are dyed in excess revolutions, are exposing or be not exposed to DNA disrupting agent Zorubicin (Dox, their cell cycle spectrum of check under situation Fig. 5).Under the situation that lacks Dox, the cell cycle of pRS-p53 cell spectrum obviously is not different from the cell cycle spectrum of pRS cell.The transient transfection of CHEK1siRNA does not have influence not exist the cell cycle under the Dox situation to compose yet.But under the situation that Dox exists, as desired to the cell of expressing functional TP53, the pRS cells transfected has shown G1 and G2/M stagnates.The excess revolutions of CHEK1siRNA is dyed and has been caused crossing the G2 outpost of the tax office, and increases at the cell number of G1 blocking-up.Because cell keeps the TP53 function, they were stagnated and are not got back to the S phase in the G1 phase.

On the contrary, the pRS-p53 cell has lost the ability of stagnating at G1, and principal reaction handles and stagnate in the G2 phase in Dox, and this is consistent in the effect at the G1 outpost of the tax office with TP53.Cell cycle spectrum by the existing pRS-p53 cell of lucsiRNA excess revolutions hair dyeing does not change (Fig. 5).LucsiRNA even the part that does not cause TP53 to react are recovered (and the corresponding increase at G1 peak), show that this siRNA does not almost have to produce the competitive inhibition of and phenotype reticent to TP53.Therefore, estimate not exist CHEK1siRNA to gather the competitive inhibition of the TP53 silence that causes.In fact, react on Dox and handle, revealed remarkable change with the pRS-p53 cell of CHEK1 transient transfection at their cell cycle stave, the cell proportion in the S phase significantly increases, and has the DNA that inferior G1 (dead cell) measures.HCT116 cell kind at pRS and pRS-p53 stable transfection has also been observed similar discovery.Therefore, destroying in the time of by the G2 outpost of the tax office of the G1 outpost of the tax office of TP53 mediation and CHEK1 mediation, is lethal to the TP53-tumour cell, but is not lethal to the TP53+ tumour cell.

The siRNA of transfection is the silence that causes of the shRNA of competitive inhibition stably express not, and this discovery is unexpected.At present not clear why competitive intersection of siRNA suppresses reticent, and shRNA and siRNA are then really not so.This may point out, and this RNA of two types different step on biological chemistry enters the RNAi approach.

Figure 15 A-C shows the result of the reticent pair cell of CHEK1 to the influence of DNA destructive susceptibility.It is responsive that 15A:CHEK1 silence/inhibition makes the HeLa cell destroy DNA.It is responsive that 15B:CHEK1 silence/inhibition makes the p53-A549 cell destroy DNA.The 15C:CHEK1 silence does not make the HREP cell to the Zorubicin sensitivity.

6.3. embodiment 3: the gene that strengthens or weaken the cell killing that the DNA disrupting agent causes

Present embodiment has illustrated a kind of semi-automatic siRNA screening that is used to identify the gene that strengthens or weaken the cell killing that the DNA disrupting agent causes.Semi-automatic platform makes it possible to that ((loss-of-function) RNAi that siRNA ' s) carries out loss of function screens with siRNA.The toughener that the library of the siRNA of the about surely 800 kinds of Human genomes of target is used for identification of dna disrupting agent Zorubicin (Dox), camptothecine (Campto) and cis-platinum (Cis).In each screening, identified many genes (" hitting thing "), its destruction makes the cell killing sensitivity of cell to chemotherapeutics.(referring to Table III A-C).These some (as WEE1) promptings of hitting in the thing strengthen the active new target of chemotherapeutics commonly used; Other hits thing, and (BRCA1, BRCA2) prompting is used for the new therapy that the cancer that the sudden change of these genes causes is determined in heredity.

In order in the human cell, to carry out genescreen, set up the library of siRNA duplex.The about surely 800 kinds of genes of a kind of form target in this library, every kind of gene adopts three kinds of siRNA.This library extends to about surely 2, the 000 kinds of genes of target, extends further to fixed＞7, the 000 kind of gene (2-3 kind siRNA/ gene) of target.This library comprises the siRNA of the fixed gene from " can be used as (druggable) genome of medicine " of target (using gene or the gene family of small molecules small molecules as medicine promptly).This library also comprises the siRNA of the fixed gene from " film group " (membranin) of target, to promote to identify the potential target of treatment antibody.Table III A-C has listed the sequence of the part of the siRNA that uses among this embodiment.In order to promote to carry out extensive siRNA screening, developed semi-automatic platform with this library.Before transfection, merge the isogenic three kinds of different siRNA of target phasing (total siRNA concentration is 100nM).Can by a people in less than 4 hours time with double complete library transfection in cell.Each siRNA set is checked 2-4 time in single experiment usually, and each experiment is repeated 2 times by Different Individual usually at least.Not on the same day or the screening undertaken by different people reached the repeatability that realizes.

The purpose of screening is to identify to make cell to cancer chemotherapeutic agent Dox, Campto commonly used and the target of Cis sensitivity.Dox (adriamycin) suppresses the activity of topoisomerase II (TopoII).TopoII mainly works in G2 and the M phase of cell cycle, and to untiing dna structure in case carry out chromosomal correct packing with separate very important.Campto suppresses topoisomerase I (TopoI).TopoI worked in the S phase, to discharge the twisting stress of advancing archaeal dna polymerase mixture.Campto is added cell in duplicating, cause replication fork to pause and the DNA splitting of chain.Cis causes dna adduct and chain crosslinked.Cis and Campto handle and cause replication fork to be stagnated and possible replication fork fracture, cause dsDNA fracture and necrocytosis.

The elementary screening of carrying out with each reagent is to carry out in the HeLa of TP53 defective cell.Gather transfection HeLa cell with siRNA, add medicine after 4 hours.Carry out the every kind drug concentrations of preliminary experiment to determine to use; Usually this is to cause the needed amount of 10%-20% growth-inhibiting (EC10 or EC20).Assessment in 72 hours after transfection+/-growth of the cell of medicine.

The structure of screening with Cis is shown among Fig. 6.Table II A shows the sensitization multiple that causes by the cis-platinum that on average surpasses the cis concentration of 400ng/ml and 500ng/ml.The figure shows under the situation that does not have medicine with the growth per-cent (log conversions) (X-axis) of siRNA set cells transfected and had growth per-cent (Y-axis) under the situation of medicine.Knocking out of some gene make to the pharmacological agent sensitization, its below diagonal lines, and some gene knock out the resistance of mediation to medicine, it is more than diagonal lines.The siRNA set that target is decided BRCA2 causes＞10 times sensitization to Cis.The siRNA of BRCA1 set causes＞3 times sensitization.The siRNA that target is decided kinases WEE1 and EPHB3 also cause to Cis＞3 times sensitization.15 kinds of genes cause＞2 times sensitization altogether.In similar screening, in each Dox and Campto screening, identified about 50 kinds cause to medicine＞gene (referring to Table II b-C) of 2 times sensitization.Overlapping hereinafter between heterogeneic group discussed.

The design that is important to note that this screening is in order to find the toughener of pharmaceutical activity.Because the drug level cell growth of using produces considerably less effect, the co-inhibitor of medicine also cell growth produces considerably less effect.Like this, as expected, we have observed considerably less gene, and the destruction of described gene has been prevented pharmaceutical activity.A significant exception is the siRNA that target is decided polo sample kinases PLK, and its activity in the presence of Cis is lower.This may reflect that DNA destroys and PLK destroys the fact that causes cell cycle arrest.When cell cycle arrest had been induced in current a kind of treatment, the curative effect of a kind of treatment in back was lower.

Cause overlapping between the gene of different pharmaceutical sensitization in order to show, we compared the cell growth of different reagent-/+ratio (Fig. 7) of medicine (sensitization multiple).Relatively cause finding the kinase whose siRNA of some gene such as WEE1, make the kill and wound sensitivity of cell two kinds of reagent to the Dox sensitization with to the gene (Fig. 7, left side) of Cis sensitization.On the contrary, the siRNA that only decides breast cancer susceptibility gene BRCA2 with target has observed the strong sensitization (＞10 times) that cell kills and wounds Cis.Relatively cause to the Campto sensitization with to the gene (Fig. 2, right side) of Cis sensitization, find the highest gene (BRCA2, BRCA1, EPHB3, WEE1 and ELK1) of scoring identical when treating with two kinds.

WEE1 destroys the observations prompting that causes all three kinds of reagent sensitizations, and this kinases is regulated the total DNA of all reagent is destroyed reaction.In the biological chemistry, human WEE1 avoids tenuigenin activated CDC2 kinases by the protection nucleus and destroys, and coordinates the transformation (Heald et al., 1993, Cell 74:463-474) between dna replication dna and the mitotic division.Other research prompting WEE1 repairs the composition at the outpost of the tax office at the DNA that the G2 phase of cell cycle works.Because most of human tumors are TP53 defectives, they lack the outpost of the tax office that the main TP53 that works in the G1 phase regulates, and therefore more depend on other outpost of the tax office (that is, they have normal pass card redundancy) than the healthy tissues of expressing TP53.Consider together to obtain data, show that WEE1 is provided for treating TP53 defective tumor treatment target, the survival of described tumour depends on G2 outpost of the tax office integrity.In fact, reported the radiosensitizer (that is, make cell to radiation inductive necrocytosis sensitivity) of the micromolecular inhibitor of WEE1, but the sensitization that this compound causes is moderate (Wang et al. as the TP53 deficient cells, 2001, Cancer Res.61:8211-7).Checked " hitting thing " ability that sensitized cell kills and wounds in other scope from these screenings in the function of the tumour cell outpost of the tax office: for example, by the cell of other tumor type and coupling+/-use other DNA disrupting agent in the TP53 function.

Consistent to the overlapping mechanism of action in the gene of Cis and Campto sensitization with these medicines.They all target decide the S phase, and the progress of replication fork is paused, cause the formation of dsRNA fracture material.On the contrary, Dox mainly works in the G2/M phase of cell cycle.Like this, may represent sensitization by BRCA1 and BRCA2 to the sensitization of Campto and Cis based on S phase specific mechanism.These results and the data consistent (D ' Andrea et al., 2003, Nat Rev Cancer3:23-34) that the research of the effect of BRCA1 and BRCA2 in the DNA destruction approach is obtained.In fact, now known these two kinds of genes all work in the DNA of the gene mediated relevant with congenital panhemocytopenia reparation approach; BRCA2 is identical with the FANCD1 of one of these genes.The cell that carries the defective in the BRCA approach has the susceptibility (Taniguchi et al., 2003, Nat Med.9:568-74) of increase to Cis.At present, the cancer patients with BRCA sudden change does not accept the treatment that target is decided their hereditary defect, but is making great efforts to detect platinum medicine (Couzin, 2003, Science 302:592) in these patients.

Consider together, these Notes of Key Datas siRNA Screening and Identification potential " reactor " colony (that is the tumour of BRCA pathway deficiency) of some DNA disrupting agent.Up to date, think that still only sub-fraction mammary gland and ovarian tumor are caused by the germ line mutation in the BRCA gene, because the tumour of distributing does not show the change in these genes usually.But nearest data show that other member's of BRCA approach inactivation of gene may the change than BRCA gene self more extensive (Marsit et al., 2004, Oncogene 23:1000-4) in the tumour of distributing.The siRNA in the bigger library of following use screens to have and helps identify other gene, and the destruction of described gene can be diagnosed the susceptibility to the DNA disrupting agent.In fact, in the siRNA library of expanding, represented a lot of known and predictions DNA-repair gene (as, comprise other congenital panhemocytopenia gene in the BRCA approach).The screening of appropriate designs also can be identified other molecule target useful to particular patient, has the BRCA pathway gene in the tumour of described particular patient and destroys.

Elementary screening is following to be carried out: screening contains the siRNA library of the siRNA of about 800 kinds of genes, the gene of adjusted cis-platinum (cis-diaminedichloroplatinum).Set (set of 3 kinds of siRNA of every kind of gene) screening library with the siRNA of 100nM (every kind of siRNA33nM).Exist or do not exist＜condition of the cis-platinum of EC25 concentration (400ng/ml) under with these siRNA transfections in the HeLa cell.

In transfection the day before yesterday, the cell that 50 microlitres are selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) grow to the about 90% cervical cancer HeLa cell (ATCC that converges in, Cat.No.CCL-2) with 450 cells/well be planted in the 384 hole tissue culturing plates (Corning, Coming, NY).For each transfection, with 20 microlitre OptiMEM (Invitrogen) with (Dharmacon, Lafayette CO) mix from 2 microlitre siRNA of 10 micromole's liquid storages.For each transfection, 20 microlitre OptiMEM are mixed with 1 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 20 microlitre OptiMEM/Oligofectamine mixtures and OptiMEM/siRNA mixture are assigned in each hole of 96 orifice plates, mix, and at room temperature incubation 15-20 minute.5 microlitre transfection mixtures are distributed in each hole of 384 orifice plates, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.4 difference 96 orifice plates that will contain different siRNA set are distributed on 384 orifice plates, and each accounts for 1/4th of 384 orifice plates.It all is to use the BioMek FX liquid processor with 96 hole distributor heads to carry out that all liquid shifts.

After 4 hours, add the DMEM/10% foetal calf serum that contains or do not contain the 2400ng/ml cis-platinum in 5 microlitres/hole, with plate incubation 68 hours under the condition of 37 ℃ and 5%CO2.Measure cell growth (referring to the 5.4.2.2 joint) with Alamar Blue assay method.AlamarBlue measures and measures cellular respiration, and with observed value measuring as viable cell number.The interior environment of proliferative cell has higher reductibility than the interior environment of non-proliferative cell.Particularly, in breeding, NADPH/NADP, FADH/FAD, the ratio of FMNH/FMN and NADH/NAF increases.These metabolic intermediates can reduce alamarBlue, therefore, can be used to monitor cell proliferation.After the transfection 72 hours, from the hole, remove substratum, and with containing 10% (vol/vol) alamarBlue reagent (Biosource International Inc., Camarillo, CA) and the DMEM/10% foetal calf serum (Invitrogen) of the 1M Hepes damping fluid tissue culture reagent (Invitrogen) of 0.001 volume replace.With plate 37 ℃ of incubations 2 hours, adopt Softmax Pro 3.1.2 software (Molecular Devices) at GeminiEM microtitration plate reader (Molecular Devices then, Sunnyvale, CA) on, excite with the 590nm emitted fluorescence by 545nm and to read plate.The relative fluorescence unit that gathers the hole of transfections from different siRNA deducts the not relative fluorescence unit in celliferous hole, to determine to be higher than the relative fluorescence unit of background level.The relative fluorescence unit in hole that exists or relative fluorescence unit and the target in the hole of siRNA set transfection decided the siRNA transfection of luciferase when not having cis-platinum is compared.Exist or when not having cis-platinum the relative fluorescence unit of luciferase siRNA cells transfected be considered to 100%.% growth with respect to luciferase by will not have medicine the time is plotted on X-axis, and the % growth with respect to luciferase when having medicine is plotted on Y-axis, has made comparison diagram.

Carried out secondary screens with HeLa cell, A549-pRS cell and A549-C7 cell.With the set (set of 3 kinds of siRNA of every kind of gene) of the siRNA of 100nM (every kind of siRNA 33nM), or with the single siRNA transfectional cell of 100nM.Under the condition that has or do not exist various concentration DNA disrupting agents with these siRNA transfections in the HeLa cell.The concentration of every kind of reagent is as follows: for the HeLa cell, and Zorubicin (10nM), camptothecine (6nM), cis-platinum (500ng/ml); For other clone, Zorubicin (200nM), camptothecine (200nM), cis-platinum (4ug/ml).

Following siRNA:WEE1 set, EPHB3 set, CHUK set, BRCA1 set, BRCA2 set and STK6 have been used.The sequence of the siRNA that uses is listed in Table III A.

Be performed as follows the siRNA transfection: in transfection the day before yesterday, the clone that 2000 microlitres are selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) grow to the about 90% cervical cancer HeLa cell (ATCC that converges in, Cat.No.CCL-2) be planted in the 6 hole tissue culturing plates with 45,000 cells/well.For each transfection, with 70 microlitre OptiMEM (Invitrogen) with (Dharmacon, Lafayette CO) mix from 5 microlitre siRNA of 20 micromole's liquid storages.For each transfection, 20 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 25 microlitre OptiMEM/Oligofectamine mixtures are mixed with 75 microlitre OptiMEM/siRNA mixtures, and at room temperature incubation 15-20 minute.100 microlitre transfection mixtures are distributed in each hole of 6 orifice plates, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.

After 4 hours, the DMEM/10% foetal calf serum that contains or do not contain the DNA disrupting agent in 100 microlitres/hole is added each hole, to reach the final concentration of every kind of reagent mentioned above.With plate incubation 44 or 68 hours again under the condition of 37 ℃ and 5%CO2.Analyze cell cycle spectrum then from the sample of two time points (after the transfection 48 hours or 72 hours).

For cell cycle analysis, will be from the supernatant liquor and the cytomixis of gathering in the crops by tryptic digestion in each hole.Then with 1200rpm with centrifugal 5 minutes of mixture.With 70% ice-cold ethanol with about 30 minutes of cell fixation.With PBS once, be suspended in 0.5ml again and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), and 37 ℃ of following incubations 30 minutes with the fixed cell washing.(Becton Dickinson) carries out flow cytometry with the FACSCalibur flow cytometer, and with FlowJo software (TreeStar, Inc) analytical data.Measure necrocytosis with inferior G1 cell mass.If (siRNA+DMSO) summation of the inferior G1 cell mass of sample and (Luc+ medicine) sample thinks that greater than the inferior G1 cell mass of (siRNA+ medicine) sample the siRNA silence makes cell destroy sensitization to DNA.

Fig. 9-14 shows the result of secondary screens.Fig. 9 A-9C represents that the silence of WEE1 makes the HeLa cell destroy responsive to Dox, Campto and Cis inductive DNA.Fig. 9 D-9I shows that the silence of WEE1 makes the p53-A549 cell destroy sensitivity to Dox, Campto and Cis inductive DNA, but it is responsive that the p53+A549 cell is destroyed described DNA.Figure 10 A-10C shows that the silence of EPHB3 makes HeLa cell and p53-A549C7 destroy sensitivity to Dox, Campto and Cis inductive DNA, makes the p53+A549pRS cell lower to the degree of Dox, Campto and Cis inductive DNA destruction sensitivity.Figure 11 A-11C shows that the STK6 silence makes HeLa cell and p53-A549C7 destroy sensitivity to Dox, Campto and Cis inductive DNA, makes the p53+A549pRS cell lower to the degree of Dox, Campto and Cis inductive DNA destruction sensitivity.Figure 12 A-12C shows that the silence of BRCA1 makes HeLa cell and p53-A549C7 cell destroy responsive to Dox, Campto and Cis inductive DNA.It is responsive that the silence of BRCA also makes the p53+A549pRS cell destroy Cis inductive DNA with lower degree, but do not make the p53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.Figure 13 A-13B shows that the silence of BRCA2 makes HeLa cell and p53-A549C7 cell destroy responsive to Dox, Campto and Cis inductive DNA.It is responsive that Figure 13 C shows that the silence of BRCA makes the p53+A549pRS cell destroy Cis inductive DNA with lower degree, but do not make the p53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.Figure 14 A-14B shows that the silence of CHUK makes the HeLa cell destroy responsive to Dox, Campto and Cis inductive DNA.Figure 14 C shows that the silence of CHUK makes the p53-A549pRS cell destroy responsive to Campto and Cis inductive DNA.Figure 14 D shows that the silence of CHUK does not make the 53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.

The average sensitization multiple that Table II A cis-platinum causes

Gene I		The gene title	Average sensitization multiple	Standard deviation
					1	2514	PLK	0.302987553	0.122442
2	3099	PLK	0.344442634	0.157221
					3	3433	PLK	0.415618617	0.142888
4	3266	PLK	0.471258534	0.273419
					5	3006	PLK	0.573026377	0.295022
6	3534	PLK	0.580135373	0.403069
					7	3806	C10orf3	0.581678284	0.122098
8	3322	CCNA2	0.603615299	0.027899
					9	3805	C20orf1	0.618083836	0.081029
10	3423	NM_006101	0.640054878	0.131981
					11	3464	INSR	0.67184541	0.043498
12	3722	TLK2	0.680201667	0.164793
					13	3731	CSNK1E	0.70971928	0.169767
14	3261	ERBB2	0.721804997	0.095466
					15	3093	PIK3CG	0.730517635	0.16341
16	3391	PLK	0.73566872	0.438713
					17	3813	ANLN	0.742286686	0.076826
18	3687	CAMK4	0.763785182	0.078326
					19	3838	PRKAA2	0.768128477	0.098461
20	2702	2702	0.77422078	0.032982

21	3435	FLT3	0.786069641	0.033061
					22	3740	STK35	0.786251834	0.241352
23	3826	NM_015694	0.78668619	0.158833
					24	3113	CNK	0.789751097	0.074976
25	3648	CLK1	0.795962486	0.119858
					26	3397	BUB3	0.798897309	0.041819
27	2982	CDC2L2	0.803290264	0.28261
					28	2975	NEK4	0.804972926	0.092313
29	3003	FER	0.806761229	0.283308
					30	3776	NOTCH2	0.807626974	0.090463
31	3600	RRM2B	0.807791139	0.116058
					32	3303	CDKN2D	0.808236038	0.106543
33	3536	PIK3C3	0.811623871	0.072924
					34	3491	PRKCE	0.818554314	0.081903
35	3181	ST5	0.820227877	0.105561
					36	3812	CDCA8	0.825194175	0.149709
37	3525	NOTCH4	0.826075824	0.135465
					38	3182	MYCN	0.826997754	0.074996
39	2992	PRKR	0.83026411	0.107682
					40	2972	KSR	0.840737073	0.220722
41	3359	TUBA1	0.841656288	0.176344
					42	3183	NM_005200	0.843755002	0.126232
43	2961	PIM1	0.846814316	0.1791
					44	3814	HMMR	0.848584565	0.089675
45	3326	CCT7	0.850805908	0.139648
					46	3819	TACC3	0.851051224	0.151449
47	3495	FGFR2	0.851658058	0.169414
					48	2952	PRKG1	0.853083744	0.103483
49	3680	CLK3	0.853111421	0.029348
					50	3650	NM_025195	0.855769333	0.097938
51	3635	STAT1	0.856732819	0.045221
					52	3487	MAP2K3	0.858609643	0.046727
53	3831	CLSPN	0.865300447	0.043122
					54	3416	IKBKE	0.868770694	0.033925
55	3693	NEK9	0.871865115	0.272749
					56	3686	MAP3K8	0.872321606	0.276021
57	3677	HCK	0.874242862	0.099478
					58	3509	KIF21A	0.876152348	0.070276
59	3666	PAK6	0.877347139	0.070142
					60	3563	RAB3A	0.877392452	0.07511
61	2993	SRMS	0.877914429	0.052743
					62	3658	STK18	0.884409716	0.022945
63	3153	RB1	0.884802012	0.066909
					64	3000	BMX	0.88790935	0.05788
65	3784	MAPK8	0.888444434	0.124134
					66	3503	EGR1	0.8888158	0.172111
67	3578	RREB1	0.889406356	0.126074
					68	3085	KIF5C	0.889747874	0.062749
69	3431	NM_018454	0.893082893	0.124062
					70	2954	ROCK2	0.893933798	0.055935
71	2922	NM_004783	0.89487587	0.052019
					72	3631	WISP2	0.895799222	0.04132

73	3752	CCNB3	0.895903064	0.014712
					74	3808	CKAP2	0.897429532	0.077036
75	3399	HSPCB	0.898588123	0.283379
					76	3251	ABL1	0.899747173	0.09061
77	3695	PRKAA1	0.899926191	0.099839
					78	3319	CCND1	0.901342596	0.14162
79	3786	FRAP1	0.901481586	0.064389
					80	2964	RIPK2	0.901658094	0.057156
81	3179	PDGFB	0.902358454	0.054703
					82	2987	RNASEL	0.90485908	0.109916
83	3086	KIF11	0.905925473	0.044166
					84	3610	LEF1	0.906026445	0.269465
85	3798	ACTR2	0.9086166	0.162743
					86	3088	KIF13B	0.912159346	0.09222
87	3332	CDC5L	0.912625936	0.141471
					88	3711	LIMK1	0.912891621	0.150911
89	3775	NOTCH1	0.914649314	0.049686
					90	3743	RAGE	0.915875434	0.062887
91	3410	RPS27	0.916611322	0.14842
					92	3403	AURKC	0.917162845	0.112884
93	3197	ARHB	0.917549671	0.07581
					94	3145	C20orf23	0.918517448	0.040236
95	2980	RIPK1	0.918693241	0.035801
					96	3646	NM_005781	0.919629184	0.074213
97	3256	CDC2L1	0.920311861	0.161437
					98	3171	VHL	0.921197139	0.154964
99	3661	FGR	0.921903863	0.062718
					100	2978	AB067470	0.922713135	0.058126
101	2983	GUCY2C	0.922891001	0.132499
					102	3557	JUND	0.923386231	0.212516
103	3573	NM_016848	0.924255509	0.025747
					104	3783	KRAS2	0.924335869	0.031975
105	3833	ATR	0.925151796	0.036459
					106	3762	MCC	0.926766797	0.063215
107	2934	IRAK2	0.927137542	0.090048
					108	3311	CDK10	0.927487493	0.197303
109	3230	MAP2K1	0.929528292	0.087866
					110	3461	KIT	0.929864607	0.065105
111	3581	RASGRP1	0.930046334	0.085936
					112	3782	SOS1	0.93078276	0.086957
113	3348	DCK	0.932934579	0.140927
					114	3518	NFKB1	0.933538042	0.254776
115	3692	AB007941	0.934031479	0.122891
					116	2936	SGKL	0.935268856	0.12869
117	3788	PRKCE	0.935825459	0.100437
					118	3791	NM_005200	0.937373151	0.124551
119	3827	NM_018123	0.938752687	0.120885
					120	3343	CENPJ	0.939276361	0.15064
121	3413	KIF23	0.940719223	0.224476
					122	3540	PPP2CB	0.940825549	0.07786
123	3559	RAP1GDS1	0.941186098	0.092318
					124	2943	DYRK2	0.941751587	0.079768

125	3090	KIF3C	0.942994713	0.043187
					126	3306	CDC14A	0.943159212	0.105314
127	3572	RASA3	0.943756386	0.044924
					128	3822	GTSE1	0.944755556	0.2332
129	3351	ESR1	0.944920378	0.153622
					130	3258	MOS	0.9460337	0.090205
131	3601	POLE	0.94708241	0.126731
					132	2960	LYN	0.947322877	0.19148
133	3828	KIF20A	0.950773558	0.183938
					134	3778	VHL	0.951938861	0.232481
135	3196	ARHI	0.952842248	0.058681
					136	3566	JUN	0.95294025	0.127285
137	3240	MAPK12	0.953564717	0.071586
					138	3184	TSG101	0.954002138	0.04823
139	3714	NM_013355	0.954197885	0.12488
					140	3364	HPRT1	0.954414394	0.271771
141	3685	LTK	0.954443302	0.285398
					142	3751	BCR	0.954467451	0.121004
143	3434	DDX6	0.954790843	0.082973
					144	3298	CCNE1	0.955113281	0.080149
145	3449	TBK1	0.955301632	0.018969
					146	3795	NR4A2	0.955557277	0.096686
147	3739	NM_017886	0.955581637	0.103771
					148	3471	MAPK10	0.956705519	0.068765
149	3139	XM_095827	0.956993628	0.217327
					150	3545	IRS2	0.957861116	0.058638
151	2985	MKNK1	0.958784274	0.02755
					152	3618	DVL2	0.958860428	0.145917
153	3726	MAPKAPK2	0.95922853	0.071282
					154	3678	PFTK1	0.960709464	0.043435
155	3821	ASPM	0.961220945	0.129044
					156	3163	THRA	0.96204376	0.138031
157	3101	MAPK14	0.962194967	0.089772
					158	3561	FOS	0.96220097	0.038394
159	3133	XM_168069	0.962355545	0.075119
					160	3443	EPS8	0.962670509	0.080284
161	3117	ATM	0.963448158	0.17684
					162	3401	HDAC1	0.963594921	0.053087
163	3799	ACTR3	0.96385153	0.106281
					164	3733	MYLK2	0.96390586	0.071956
165	3801	PSEN1	0.96432309	0.133399
					166	3716	ULK1	0.964714374	0.172756
167	2977	RIPK3	0.965321488	0.288006
					168	3571	VAV1	0.966085791	0.040696
169	2946	NM_017719	0.966726854	0.070416
					170	3459	EGFR	0.968475197	0.03989
171	3835	CHEK2	0.968492479	0.077394
					172	3125	NM_031217	0.968705878	0.158815
173	3308	CDKN2B	0.970454697	0.030316
					174	3458	ARAF1	0.972126514	0.150383
175	3162	MADH2	0.97228749	0.077251
					176	2949	MYO3B	0.973636618	0.06916

177	3664	STK17A	0.975343312	0.06811
					178	3488	AURKB	0.975742132	0.178321
179	3112	KNSL7	0.976665103	0.157911
					180	3485	DHX8	0.978053596	0.073262
181	3809	CDCA3	0.979002265	0.231181
					182	3161	WT1	0.979221693	0.114838
183	3513	ROS1	0.979271577	0.121589
					184	3185	VCAM1	0.979438257	0.069759
185	3553	CKS1B	0.979465469	0.05555
					186	3763	NM_016231	0.980990574	0.123555
187	3245	AXL	0.981022783	0.078724
					188	3334	CUL4B	0.981485893	0.048462
189	3193	FGF3	0.981515057	0.075982
					190	3335	CDK5R2	0.983188137	0.095535
191	3455	MAP2K4	0.984383299	0.095921
					192	2925	FYN	0.984597535	0.177611
193	3215	MAD2L1	0.984674289	0.166817
					194	3519	NTRK1	0.985625526	0.225903
195	2541	2541	0.985658529	0.02934
					196	3109	KIF1C	0.985891536	0.162583
197	3792	ARHGEF1	0.985986394	0.150503
					198	3374	POLR2A	0.986875398	0.174675
199	3362	NR3C1	0.98711375	0.09249
					200	3231	ILK	0.987500124	0.068942
201	3166	PMS1	0.987593476	0.040016
					202	3703	AK024504	0.988238947	0.078314
203	3707	TXK	0.98900485	0.138186
					204	3323	CDK5R1	0.989595604	0.176376
205	3180	CD44	0.990121058	0.090413
					206	3630	WISP3	0.990225627	0.071631
207	3576	GRAP	0.990505346	0.120959
					208	3800	CHFR	0.99369692	0.117429
209	3142	KIF25	0.993932342	0.044087
					210	3160	TACSTD1	0.994447265	0.128513
211	3497	EPHA8	0.99446771	0.015206
					212	3757	CLK4	0.995683284	0.166859
213	3645	CASK	0.996395727	0.07959
					214	3357	PRIM2A	0.998092371	0.117247
215	3594	RAP2A	0.99814842	0.142818
					216	3796	ARHGEF6	0.998577367	0.091413
217	3767	FZD3	0.99921132	0.096395
					218	3715	CDC42BPA	0.999524848	0.196389
219	2938	ALS2CR7	0.99966653	0.007718
					220	3419	RFC4	0.999756476	0.079342
221	63	M15077	1	0
					222	3672	SYK	1.000094306	0.028316
223	3832	ATM	1.00019002	0.091546
					224	3627	CTNNA1	1.000291459	0.215453
225	2984	EPHB6	1.000603948	0.098044
					226	3200	REL	1.000616585	0.104464
227	3492	PRKCQ	1.000785085	0.103181
					228	3478	EPHA2	1.001756995	0.101444

229	3539	PLCG2	1.002008086	0.072305
					230	3378	NM_006009	1.002912861	0.019886
231	3381	POLR2B	1.003653073	0.021542
					232	3452	JAK1	1.00410017	0.246916
233	2926	AF172264	1.0043601	0.195291
					234	3641	TYRO3	1.005062954	0.13144
235	3750	CAMK2A	1.005879519	0.197981
					236	3595	FEN1	1.006107713	0.1559
237	3375	AHCY	1.006847357	0.098914
					238	3367	DHFR	1.007697409	0.048476
239	3555	RASA1	1.007907357	0.060107
					240	3246	RPS6KB1	1.008295705	0.098199
241	3551	STAT3	1.008697776	0.069559
					242	3708	RPS6KC1	1.008738004	0.158539
243	3820	NM_018410	1.008803441	0.00423
					244	3548	RAC1	1.009000664	0.10905
245	3527	DTX2	1.00940399	0.082767
					246	3339	CCNB2	1.009625325	0.321434
247	3226	RBX1	1.01029159	0.235969
					248	3473	DAPK1	1.010335394	0.065266
249	3469	AAK1	1.011653085	0.153819
					250	3517	MYC	1.011855757	0.088032
251	3005	MERTK	1.011910266	0.139112
					252	3294	CCNF	1.01355402	0.151217
253	3392	BIRC5	1.014018575	0.147292
					254	3533	HES7	1.016868954	0.209244
255	3524	NOTCH3	1.017285472	0.068877
					256	3587	VAV3	1.018129173	0.062737
257	3425	DLG7	1.018264827	0.037325
					258	3674	CSNK1D	1.018650699	0.087521
259	3380	TUBG2	1.019248432	0.027697
					260	3486	RPS6KA3	1.019985527	0.050031
261	3746	HUNK	1.020779918	0.082372
					262	3535	SKP2	1.021142953	0.100064
263	3797	ARHGEF9	1.021635562	0.137783
					264	2969	NM_014916	1.021887811	0.080467
265	3460	CSK	1.022085366	0.135805
					266	3132	KIF23	1.023806782	0.129496
267	2963	MAP3K11	1.0242873	0.065223
					268	3702	MAP3K13	1.024294874	0.083014
269	3382	TUBB	1.025915608	0.049937
					270	3237	CDC7	1.025994603	0.096409
271	3592	SOS2	1.026235513	0.178995
					272	3365	PRIM1	1.02653855	0.104798
273	3570	RALGDS	1.027460697	0.084873
					274	3224	FBXO5	1.029155584	0.154545
275	3585	GAB1	1.029481526	0.06077
					276	3414	HDAC7A	1.030424895	0.139587
277	3514	HRAS	1.030481671	0.09281
					278	3597	SHMT2	1.031207997	0.180827
279	3657	PCTK1	1.031839128	0.067828
					280	3257	IGF1R	1.03192729	0.10264

281	3773	WNT2	1.032309731	0.174004
					282	3625	CTBP2	1.032538009	0.159078
283	3302	CDK8	1.032760545	0.077771
					284	3409	TTK	1.033113517	0.089383
285	3465	EPHA1	1.033516487	0.127809
					286	3705	NM_012119	1.033751364	0.107948
287	2966	NM_033266	1.035012335	0.097641
					288	2999	FES	1.035558582	0.12725
289	3474	CSNK2A1	1.036151218	0.085057
					290	3824	MAPRE3	1.036197838	0.092706
291	3094	KIF3A	1.036464942	0.119921
					292	3769	PLAU	1.037390211	0.064893
293	3213	NM_016238	1.037745909	0.138786
					294	2950	NEK6	1.038291854	0.149776
295	3815	MAPRE2	1.038305947	0.217709
					296	3543	PDK2	1.038311221	0.197679
297	3437	FGFR1	1.0383269	0.283643
					298	3542	PPP2CA	1.039357671	0.194352
299	3511	XM_168069	1.039370913	0.155262
					300	3002	CRKL	1.039983971	0.101129
301	3398	HDAC11	1.041663934	0.099406
					302	3675	ADRBK1	1.041741459	0.084419
303	3623	CTNND1	1.042210238	0.105978
					304	3268	CDC25C	1.042762357	0.02726
305	3633	CTBP1	1.042818569	0.143171
					306	3804	NM_024322	1.042895573	0.05266
307	3526	HES6	1.043146787	0.059244
					308	2947	NM_007064	1.043456689	0.080305
309	2979	PAK2	1.043537793	0.115188
					310	2959	PIM2	1.043942064	0.050352
311	3602	MCM3	1.044071108	0.23865
					312	3665	PAK4	1.044246523	0.052921
313	3421	ORC6L	1.044825423	0.241726
					314	3745	CAMKK2	1.044966032	0.032844
315	3736	PTK7	1.045008777	0.118965
					316	3119	CDKN1B	1.045154749	0.026803
317	3643	DDR2	1.045796426	0.123748
					318	3603	POLS	1.046796283	0.090212
319	3346	CCNK	1.047737442	0.148152
					320	3438	DTR	1.04975054	0.139619
321	2942	TTN	1.050944386	0.134575
					322	2937	NM_025052	1.051593448	0.052118
323	3577	RAB2L	1.051977248	0.073992
					324	3203	ITGA5	1.052197011	0.109443
325	3599	DTYMK	1.052206896	0.147041
					326	3373	top2A	1.053946926	0.061071
327	3222	PTTG1	1.054934465	0.059734
					328	3154	MADH4	1.055367142	0.392285
329	3829	KIF2C	1.056017438	0.187684
					330	3652	PDGFRA	1.056020056	0.084537
331	2944	MARK1	1.056491568	0.161232
					332	3656	PRKCN	1.056755878	0.177943

333	3626	DVL3	1.058711269	0.19647
					334	3802	NOTCH3	1.059031918	0.117495
335	3127	MAPK1	1.059441261	0.070449
					336	3549	PIK3R2	1.059495493	0.178697
337	2935	MAPK6	1.060533709	0.075031
					338	3307	CDC6	1.060858236	0.093933
339	3260	STK11	1.061848445	0.120762
					340	3766	S100A2	1.062832073	0.174576
341	3457	BAD	1.063944791	0.07637
					342	3347	top1	1.06614481	0.169748
343	3450	MAP3K2	1.066638971	0.166869
					344	3164	MYCL1	1.066666964	0.198532
345	3412	KIF25	1.068536113	0.202887
					346	3317	CCNI	1.068966464	0.126188
347	3550	PLCG1	1.069052894	0.123064
					348	3668	DAPK3	1.069120278	0.16697
349	3454	FLT4	1.06985122	0.129807
					350	3394	HDAC6	1.070168765	0.050617
351	3122	ATSV	1.071291871	0.126675
					352	3169	NME1	1.071353382	0.062921
353	3342	CCNT1	1.07208287	0.030624
					354	3523	NOTCH2	1.072235801	0.096808
355	3591	RALB	1.072637191	0.131285
					356	2970	AATK	1.073460682	0.079116
357	3593	VAV2	1.073649235	0.087372
					358	3489	SRC	1.074621049	0.096347
359	3363	GART	1.076380891	0.07919
					360	3097	KIF20A	1.077741628	0.065192
361	3494	MAPK4	1.077922895	0.072549
					362	3114	PIK3CD	1.078095752	0.118845
363	2976	NEK7	1.078108286	0.543136
					364	3352	NR3C2	1.078524745	0.200714
365	3115	MDM2	1.079408163	0.109166
					366	3108	KIF22	1.080326814	0.089686
367	2973	NEK1	1.080546527	0.210334
					368	3219	CENPC1	1.080637703	0.211586
369	3583	JUNB	1.080828682	0.061182
					370	3476	PRKCD	1.081421932	0.063705
371	3717	NTRK2	1.08184551	0.179359
					372	3760	CDKL5	1.082031957	0.122857
373	3744	PRKWNK4	1.082821695	0.041089
					374	3147	CDKN2A	1.083174768	0.142556
375	3170	BLM	1.083396707	0.087103
					376	3390	NM_080925	1.083814073	0.187306
377	3691	NM_024046	1.084007951	0.455202
					378	3682	DYRK1A	1.085383077	0.13164
379	3338	CUL4A	1.085696166	0.113752
					380	3445	BMPR1A	1.08653048	0.217388
381	3639	STAT6	1.087172241	0.240711
					382	3683	NM_003138	1.087765627	0.107482
383	3694	STK38	1.088925769	0.15309
					384	3228	CDC27	1.089546461	0.230438

385	2923	ERN1	1.090052682	0.160503
					386	3366	TYMS	1.090784989	0.157841
387	3816	NM_017769	1.090876067	0.170619
					388	3107	KIF2	1.091300875	0.082185
389	3262	LATS1	1.09148938	0.058919
					390	3188	PMS2	1.092050213	0.140727
391	3498	CSNK1A1	1.092706943	0.059983
					392	3293	CDC25A	1.092986402	0.099227
393	3721	ANKRD3	1.093127467	0.114101
					394	3793	MAPRE1	1.093414458	0.107517
395	3305	CDC2L5	1.095069991	0.058969
					396	3647	YES1	1.095220118	0.439175
397	3324	CUL5	1.095253758	0.109464
					398	2965	NM_014720	1.095861428	0.295852
399	3300	CDC14B	1.095900812	0.053276
					400	3296	CDKN2C	1.096728587	0.06043
401	3724	EPHA7	1.096779937	0.20814
					402	3165	FGF2	1.099204865	0.052442
403	2928	IRAK1	1.099544846	0.11705
					404	3502	PRKCH	1.099795802	0.076493
405	3728	TIE	1.100408042	0.059759
					406	3424	EZH2	1.100414429	0.137994
407	3756	CDK5RAP2	1.10148794	0.169172
					408	2920	EIF2AK3	1.101874679	0.193517
409	3556	RAP1A	1.102603353	0.216629
					410	3214	CENPF	1.102666055	0.229565
411	3102	CKS2	1.103490084	0.276109
					412	2974	NEK11	1.103575721	0.38662
413	3297	CCT2	1.103974529	0.075386
					414	3393	HDAC2	1.104472861	0.074707
415	3568	PLD1	1.104812311	0043874
					416	3470	RPS6KA1	1.104927224	0.121509
417	3496	EIF4EBP2	1.105081332	0.026061
					418	3432	PRC1	1.105087833	0.109514
419	3446	PRKCG	1.105375817	0.11356
					420	3512	TGFBR1	1.106970197	0.08351
421	3749	NM_139021	1.107176607	0.060956
					422	3807	SPAG5	1.107200152	0.190375
423	3579	PDZGEF2	1.108384492	0.106374
					424	3422	SMC4L1	1.108967343	0.168462
425	3830	NM_013296	1.109620231	0.124287
					426	3537	EIF4EBP1	1.110833969	0.090069
427	3684	STK38L	1.110835442	0.127517
					428	3681	SRPK1	1.11126095	0.138319
429	2990	NM_015112	1.111540967	0.290052
					430	3605	FZD4	1.111605705	0.110001
431	3477	FGFR4	1.111898761	0.065007
					432	3490	ERBB3	1.113605654	0.088278
433	3575	LATS2	1.113869957	0.121325
					434	3755	CDKL3	1.114362934	0.239022
435	3205	NM_139286	1.114649942	0.243935
					436	3105	BUB1	1.114727935	0.21132

437	3389	NM_052963	1.114830338	0.092164
					438	3110	KIF13A	1.116195509	0.073039
439	3608	MAP3K7IP1	1.117324513	0.193266
					440	2957	TYK2	1.11788043	0.120323
441	2996	MAPK3	1.117972689	0.307163
					442	3628	CTNNBL1	1.118548429	0.092234
443	3624	CTNNB1	1.118609166	0.170984
					444	3159	RET	1.118867767	0.029128
445	3120	PIK3CB	1.119135316	0.222604
					446	3742	RHOK	1.119296716	0.166613
447	3136	XM_066649	1.119463101	0.130616
					448	3328	CCNC	1.119489673	0.067201
449	3199	NF2	1.119765637	0.070805
					450	3309	CCND2	1.121333431	0.146937
451	3143	NM_017596	1.121623368	0.07995
					452	3208	ZW10	1.121902285	0.144279
453	3753	CDK5	1.123427629	0.130821
					454	3001	PRKY	1.125456942	0.164937
455	3729	RYK	1.125623162	0.196578
					456	3156	MSH2	1.125991819	0.128643
457	3253	PRKCA	1.126352597	0.097687
					458	3607	TLE1	1.126388877	0.255505
459	3818	A1338451	1.126447243	0.085307
					460	3530	NOTCH1	1.127939559	0.128155
461	3141	NM_145754	1.129479267	0.026346
					462	3768	ARAF1	1.129705288	0.086972
463	3596	SHMT1	1.129818514	0.046177
					464	3653	NPR2	1.129853377	0.184709
465	3640	STAT5B	1.132589635	0.299667
					466	2924	STK25	1.13270396	0.083695
467	3356	TUBG1	1.133623741	0.248371
					468	3008	SGK2	1.135373086	0.09569
469	3499	GRB2	1.135404457	0.174583
					470	3506	XM_095827	1.135823602	0.058483
471	3770	TGFBR2	1.136061775	0.283098
					472	3441	PRKCI	1.137712494	0.174946
473	3609	FZD3	1.138082685	0.180803
					474	3370	AR	1.139336644	0.114355
475	3126	KIF3B	1.139588548	0.094914
					476	3508	KIF25	1.140573718	0.158738
477	3233	ROCK1	1.140584559	0.236383
					478	2941	DYRK3	1.142052549	0.138936
479	3336	CDC37	1.142173919	0.132765
					480	3741	RPS6KB2	1.142253082	0.114889
481	3546	INPP5D	1.142646282	0.17732
					482	3350	ADA	1.14270522	0.202027
483	3759	NM_006622	1.143528436	0.053271
					484	3149	TP53	1.144116968	0.028664
485	3310	CDC34	1.145001246	0.124753
					486	3267	CCNH	1.145203121	0.081778
487	3638	STAT5A	1.145284022	0.182015
					488	3564	RALBP1	1.145302766	0.187726

489	3360	RRM2	1.145371751	0.106232
					490	3662	LCK	1.145638747	0.091184
491	3223	NM_016263	1.145667614	0.160673
					492	3408	PIN1	1.146539359	0.100954
493	2986	ACVR2	1.146661813	0.10512
					494	3304	CCNE2	1.146795654	0.102769
495	2997	MST1R	1.147221866	0.283163
					496	3194	RARB	1.14777913	0.330433
497	3669	NTRK3	1.148222927	0.037566
					498	3616	FZD1	1.148473923	0.242876
499	3255	CDK7	1.148553587	0.16951
					500	3238	MAP3K3	1.1489897	0.067123
501	3613	DVL1	1.14901778	0.082647
					502	3614	CTNND2	1.14937005	0.187988
503	3318	CUL2	1.150267783	0.078013
					504	3644	EPHB1	1.15071896	0.123257
505	3567	SHC1	1.151587989	0.124227
					506	3116	KIF5A	1.152039039	0.280422
507	3148	LIG1	1.152183128	0.190895
					508	3765	CREBBP	1.154589409	0.128712
509	3232	KDR	1.156581097	0.11153
					510	3748	NM_016507	1.157187762	0.187551
511	3428	ECT2	1.157383105	0.171141
					512	3649	CAMK2B	1.157415503	0.051472
513	3426	TK1	1.158048559	0.161458
					514	3250	CHEK2	1.158473201	0.099737
515	3636	STAT2	1.158495567	0.161875
					516	3187	WNT7B	1.158590559	0.024217
517	3505	STK6	1.159146577	0.058438
					518	3341	APLP2	1.160801239	0.196169
519	3606	CREBBP	1.161326405	0.064695
					520	3263	CDC2	1.161570849	0.095606
521	2939	TLK1	1.163052719	0.110779
					522	3123	AKT3	1.163145874	0.306815
523	3615	FZD2	1.16322422	0.169339
					524	3688	GUCY2D	1.164321663	0.152801
525	3379	NM_032525	1.16446975	0.074802
					526	3710	GPRK2L	1.164900142	0.112446
527	3611	CTNNAL1	1.166257931	0.037335
					528	3521	MET	1.168013918	0.232923
529	3659	NM_015978	1.169523683	0.056392
					530	3582	GRAP2	1.170573492	0.055118
531	3562	RASD1	1.171229699	0.101195
					532	3723	NM_018401	1.1718512	0.239747
533	3130	FRAP1	1.172072928	0.029779
					534	3772	RPS6KB1	1.172823934	0.066518
535	3333	CCNT2	1.174235642	0.214732
					536	3501	RPS6KA2	1.175781534	0.193038
537	3803	MPHOSPH1	1.176011971	0.128864
					538	3248	JAK2	1.176020977	0.176345
539	3538	NFKB2	1.176129803	0.052353
					540	3732	CSNK2A2	1.177521611	0.231267

541	3730	TESK1	1.177904268	0.212526
					542	2989	ACVR1B	1.178578161	0.217492
543	3327	CDC45L	1.180204158	0.299357
					544	3301	CCNB1	1.180864124	0.162992
545	3092	KIF12	1.181937605	0.088708
					546	3239	CDK6	1.182157904	0.061044
547	3190	WNT4	1.182697676	0.072644
					548	3811	NM_152524	1.183191701	0.14187
549	2940	DCAMKL1	1.184491938	0.124698
					550	3761	WT1	1.184547796	0.16129
551	3439	EGR2	1.185671136	0.105284
					552	3295	CDK2AP1	1.187045536	0.206856
553	3817	NM_019013	1.187780743	0.082116
					554	3754	CDKL2	1.188123077	0.122877
555	3663	ALS2CR2	1.188246404	0.140777
					556	3718	PTK6	1.188784586	0.067781
557	3236	PTK2B	1.189818532	0.352399
					558	3475	EPHB4	1.189888477	0.105406
559	3211	BUB1B	1.189896824	0.292886
					560	3411	HIF1A	1.19069666	0.245883
561	2927	MAPK13	1.190710916	0.129633
					562	3264	CDK3	1.191042267	0.100335
563	3207	MAD1L1	1.191232546	0.092266
					564	3372	TUBA8	1.191540593	0.058385
565	3349	IMPDH1	1.191967983	0.225505
					566	3353	PGR	1.192360399	0.019529
567	3252	NEK2	1.192601635	0.282445
					568	3515	PDGFRB	1.192640873	0.057678
569	3216	CDC20	1.193040181	0.077143
					570	2971	DAPK2	1.19306626	0.085366
571	3552	PDK1	1.194218291	0.064673
					572	3823	NM_017779	1.194861064	0.138396
573	3528	TCF3	1.195851197	0.12389
					574	3201	RARA	1.19739521	0.087982
575	2945	CDC42BPB	1.19753147	0.087566
					576	3634	BTRC	1.201076339	0.175356
577	3377	NM_006088	1.202720091	0.096411
					578	3781	SRC	1.202931814	0.331139
579	3516	ARHA	1.203109256	0.159342
					580	3700	AB037782	1.204329771	0.402706
581	3699	NM_032844	1.207623266	0.248703
					582	2931	MAP4K3	1.211854633	0.149673
583	3189	MYB	1.212003569	0.117606
					584	3586	RASA2	1.212166142	0.210711
585	3836	TP53	1.212183899	0.169152
					586	3206	ANAPC5	1.213545746	0.079256
587	3701	STK10	1.214412753	0.23119
					588	3210	NM_013366	1.21450599	0.307913
589	3472	MAP3K5	1.215042561	0.128134
					590	3371	NM_006087	1.216059457	0.10804
591	3825	NM_152562	1.216108937	0.153345
					592	3106	KIF9	1.217161737	0.277445

593	3249	MAP2K6	1.217382649	0.23408
					594	3186	ETS1	1.218428895	0.125809
595	3541	PKD2	1.220374384	0.252301
					596	3654	VRK2	1.221266095	0.180133
597	3151	MLH1	1.221977195	0.100529
					598	3325	CDKN1C	1.222573895	0.183555
599	3774	CDC45L	1.223373496	0.170335
					600	3354	RRM1	1.224155502	0.163218
601	3225	NM_013367	1.226360075	0.377268
					602	3837	PRKAA1	1.226867311	0.260099
603	2930	ITK	1.227438086	0.134102
					604	3118	NM_032559	1.22825648	0.037564
605	3316	CCNA1	1.228667093	0.203935
					606	3651	VRK1	1.229029159	0.155208
607	3368	top3A	1.229032423	0.14134
					608	3376	AGA	1.231363135	0.128058
609	3735	PRKACB	1.231534849	0.092436
					610	3007	MAP3K14	1.234231675	0.17551
611	3420	NM_014109	1.234890989	0.370528
					612	3131	KIF1B	1.235185989	0.242087
613	3444	NEK3	1.23520517	0.358385
					614	2919	OSR1	1.237086236	0.106562
615	3128	AKT2	1.242407611	0.124394
					616	3810	Al278633	1.243126735	0.165137
617	3337	CDKN1A	1.244628023	0.018797
					618	3091	KIFC3	1.244757733	0.153624
619	3191	WNT2	1.244817311	0.187282
					620	3146	KIF21A	1.24572579	0.041267
621	3220	ANAPC11	1.246197987	0.17988
					622	3785	GRB2	1.248971557	0.108491
623	3195	CDH1	1.250259859	0.186152
					624	3500	SGK	1.251914788	0.059299
625	3103	PIK3CA	1.252248606	0.068043
					626	3507	NM_145754	1.252689721	0.222951
627	3565	RAB2	1.253586902	0.186552
					628	3462	TGFBR2	1.256459996	0.136016
629	3229	PRKCL1	1.256649876	0.153419
					630	3790	ERBB3	1.260353633	0.104586
631	3704	ACVR2B	1.261857433	0.075994
					632	3340	CENPH	1.262786326	0.131215
633	3598	PCNA	1.265667779	0.116032
					634	2967	NM_016653	1.266923195	0.232771
635	3725	EPHA4	1.267438993	0.19056
					636	2932	MAPKAPK3	1.268643945	0.025332
637	3167	S100A2	1.270859999	0.069296
					638	2994	MATK	1.271154735	0.128018
639	3315	CCT4	1.272355192	0.309065
					640	3344	CDKL1	1.272536383	0.273155
641	3689	BLK	1.27387895	0.218306
					642	3104	CDK4	1.276578446	0.161716
643	3604	TK2	1.277912947	0.101801
					644	3209	MAD2L2	1.278038114	0.253976

645	3554	PIK3R3	1.280284314	0.228353
					646	3218	CDC23	1.280483947	0.334381
647	3670	MAP3K10	1.280649754	0.129166
					648	3532	NM_019089	1.280872331	0.138131
649	3558	RALA	1.282343213	0.193164
					650	3440	FGFR3	1.283277949	0.278946
651	3779	CTNNA1	1.285066069	0.05853
					652	3312	CUL3	1.286086663	0.095171
653	3111	KIF5B	1.286155963	0.045454
					654	3320	CCND3	1.286427699	0.049781
655	3493	MAPK9	1.286708555	0.204254
					656	3463	TEC	1.286731353	0.116346
657	3198	ICAM1	1.287087211	0.105164
					658	2933	MAP4K5	1.288848714	0.19588
659	2995	PTK2	1.289781227	0.122006
					660	3637	STAT4	1.291478071	0.126765
661	3089	KIFC1	1.292296631	0.104185
					662	3330	CDK9	1.293189989	0.200332
663	3588	RHEB	1.295786915	0.113922
					664	3589	SOS1	1.300834959	0.028846
665	3418	CENPA	1.300851356	0.224648
					666	3314	CCNG1	1.302132075	0.167018
667	3697	CAMK2G	1.305521288	0.141582
					668	3620	AXIN2	1.306881235	0.175725
669	2921	RPS6KA5	1.307895788	0.116976
					670	3157	NF1	1.312364005	0.21979
671	3172	PLAU	1.314219765	0.221395
					672	3221	top3B	1.316767724	0.153732
673	3529	DTX1	1.317104131	0.100042
					674	3520	NRAS	1.318162379	0.337798
675	3138	KIF17	1.319021332	0.047239
					676	3466	JAK3	1.324590857	0.341923
677	3447	PRKCM	1.325852782	0.090164
					678	3396	HDAC10	1.327036067	0.095421
679	3405	HDAC8	1.328986383	0.137456
					680	2956	PRKCL2	1.329759987	0.277544
681	3771	PIK3CA	1.330927854	0.318605
					682	3100	GSK3B	1.332661843	0.172085
683	3140	XM_089006	1.334634688	0.210199
					684	3417	HDAC3	1.334739136	0.213735
685	2912	MPHOSPH1	1.335606817	0.192246
					686	3453	MAP2K2	1.337178184	0.231133
687	3777	ABL1	1.337946355	0.13381
					688	2991	NPR1	1.339668528	0.213719
689	3234	CDK2	1.341378632	0.327603
					690	3617	CTNNBIP1	1.344129969	0.172119
691	3217	NM_014885	1.346642104	0.37578
					692	3632	WISP1	1.34798621	0.267584
693	3404	PPARG	1.350608226	0.328799
					694	3834	CHEK1	1.353682807	0.177332
695	3244	PRKCZ	1.354506447	0.423569
					696	3242	PRKCB1	1.356110177	0.177856

697	2998	MAPK7	1.357915027	0.320918
					698	3227	NUMA1	1.358033567	0.336206
699	3676	MAP4K1	1.360624202	0.35665
					700	3087	PTEN	1.361043077	0.221803
701	3734	BMPR1B	1.362751745	0.19663
					702	3569	RASGRP2	1.362852713	0.083746
703	2953	MAPK11	1.367417583	0.335411
					704	3355	GUK1	1.368854888	0.121328
705	3713	PRKG2	1.370762753	0.096281
					706	3415	HSPCA	1.373102391	0.311637
707	3212	NM_022662	1.373845206	0.3643
					708	3789	ELK1	1.378293297	0.119276
709	3395	HDAC5	1.380918509	0.316118
					710	3448	NM_016231	1.382981639	0.250346
711	3737	NM_016457	1.384393078	0.35016
					712	3456	FLT1	1.387001071	0.117573
713	3696	NM_016281	1.392513247	0.179922
					714	3124	KIF4A	1.392774473	0.395931
715	3451	MAP3K4	1.39359615	0.215328
					716	3738	PRKWNK3	1.395197042	0.116947
717	3719	BMPR2	1.395676978	0.083941
					718	3429	MCM6	1.399624047	0.422475
719	3243	NM_004203	1.401278663	0.303675
					720	3660	DMPK	1.402604745	0.203011
721	3084	KIF14	1.405667444	0.022909
					722	3574	SH3KBP1	1.408096057	0.080055
723	3137	KIF26A	1.410397298	0.209271
					724	3671	STK4	1.410482157	0.306699
725	3202	MCC	1.410775773	0.285878
					726	3134	XM_170783	1.415482722	0.226917
727	3204	CDC16	1.415780291	0.221962
					728	3121	PIK3C2A	1.415950012	0.152356
729	3321	CDKN3	1.416176725	0.03826
					730	2951	AJ311798	1.416769938	0.177239
731	3504	PIK3CB	1.417592955	0.174632
					732	2955	PAK1	1.418341722	0.07362
733	3612	TCF1	1.421215206	0.099637
					734	3655	CAMK2D	1.422993988	0.227042
735	3135	XM_064050	1.42777471	0.205042
					736	3400	BCL2	1.432342001	0.20388
737	3794	WASL	1.432665996	0.093756
					738	3667	NM_016542	1.434249905	0.174478
739	3407	HDAC9	1.437540011	0.194891
					740	3430	STMN1	1.437943227	0.313077
741	3698	ADRBK2	1.440932223	0.248217
					742	3547	FOXO1A	1.461102151	0.113568
743	3265	RAF1	1.463315846	0.191102
					744	3690	PRKAA2	1.470696795	0.233827
745	3510	CDK4	1.487289818	0.089318
					746	3254	CSF1R	1.487946096	0.480379
747	3622	FZD9	1.49526423	0.086599
					748	3544	IRS1	1.495662211	0.040512

749	2948	MYO3A	1.4970851	0.081259
					750	3467	MAP2K7	1.497928287	0.281253
751	3096	AKT1	1.498269053	0.114084
					752	2968	STK17B	1.504346366	0.413498
753	3402	HDAC4	1.508119762	0.213089
					754	3764	NOTCH4	1.510551197	0.081586
755	3621	CTNNA2	1.511056295	0.111649
					756	3168	DCC	1.513409582	0.192599
757	2701	2701	1.51348211	0.083391
					758	3629	AXIN1	1.520734118	0.174178
759	3361	IMPDH2	1.523631011	0.067873
					760	3129	STK6	1.527103437	0.134504
761	3679	CLK2	1.53626119	0.44738
					762	3709	X95425	1.537827387	0.437676
763	2962	MAP4K2	1.547893113	0.329021
					764	3442	ERBB4	1.551008953	0.146803
765	3247	NM_018492	1.552802846	0.137033
					766	3720	AB002301	1.553258633	0.313891
767	3584	RASAL2	1.563405608	0.137336
					768	3299	CUL1	1.589913659	0.169909
769	3522	KRAS2	1.590661017	0.06841
					770	3590	ARHGEF2	1.597950927	0.252526
771	3406	TERT	1.600169172	0.094096
					772	3259	MAPK8	1.601990057	0.333883
773	3369	NM_007027	1.605090071	0.162172
					774	3787	FZD4	1.621497881	0.053639
775	2929	CHUK	1.646057679	0.111716
					776	3468	ABL2	1.652007602	0.180802
777	2988	FRK	1.653152871	0.298882
					778	3758	RAD51L1	1.662423293	0.135675
779	3531	NM_021170	1.666989154	0.094206
					780	3155	ATR	1.680571715	0.388687
781	3747	GSK3A	1.688637713	0.38953
					782	3144	KIF4B	1.695873891	0.467258
783	3235	CHEK1	1.697910825	0.356224
					784	3313	CCNG2	1.703651114	0.216266
785	3004	MAP3K1	1.721492222	0.376438
					786	3619	FRAT1	1.761446915	0.292031
787	3192	WNT1	1.765037748	0.394063
					788	3673	DDR1	1.770053978	0.2338
789	3358	top2B	1.800293702	0.195754
					790	2981	ALK	1.84348901	0.208338
791	2958	PRKACA	1.889142934	0.494773
					792	3152	APC	1.894694006	0.191358
793	3712	RPS6KA6	1.957145081	0.421292
					794	3436	BRAF	1.999825737	1.173208
795	3727	GPRK6	2.044605743	0.256806
					796	3780	MCM3	2.062893191	0.187038
797	3329	CDC42	2.131693629	0.483392
					798	3095	KIF2C	2.163834467	0.289685
799	3098	CENPE	2.170456559	0.120025
					800	3331	CDC25B	2.199340751	0.484716

801	3706	C20orf97	2.377822809	0.678329
					802	3580	ELK1	2.456195789	0.434043
803	3241	WEE1	2.66755235	0.625231
					804	3642	EPHB3	2.758093154	0.565256
805	3158	BRCA1	2.878071685	0.418358
					806	3150	BRCA2	11.61633698	1.101248

The average sensitization multiple that Table II B camptothecine causes

Gene I	BIOID	Gene	The sensitization multiple
				1	63	M15077	1
2	2514	PLK	0.029197
				3	2540	2540	#DIV/01
4	2541	2541	0.860453
				5	2701	2701	0.034091
6	2702	2702	0.432441
				7	3391	PLK	0.052632
8	3534	PLK	0.083815
				9	3099	PLK	0.090142
10	3006	PLK	0.09146
				11	3266	PLK	0.096774
12	3433	PLK	0.13
				13	3322	CCNA2	0.264029
14	3154	MADH4	0.361653
				15	3518	NFKB1	0.372726
16	3600	RRM2B	0.381056
				17	3184	TSG101	0.432287
18	3348	DCK	0.446467
				19	3332	CDC5L	0.451264
20	3812	CDCA8	0.453177
				21	3423	NM_006101	0.478261
22	3464	INSR	0.480578
				23	2961	PIM1	0.51581
24	3661	FGR	0.517647
				25	3171	VHL	0.524194
26	3809	CDCA3	0.529046
				27	3525	NOTCH4	0.534058
28	3093	PIK3CG	0.557692
				29	3740	STK35	0.55782
30	3435	FLT3	0.56
				31	3805	C20orf1	0.564035
32	3219	CENPC1	0.575465
				33	3003	FER	0.579832
34	3183	NM_005200	0.580153
				35	3374	POLR2A	0.583796
36	3601	POLE	0.588331
				37	3112	KNSL7	0.597685
38	3489	SRC	0.606833
				39	3478	EPHA2	0.608258
40	3422	SMC4L1	0.608696
				41	3357	PRIM2A	0.611218
42	3262	LATS1	0.613321
				43	2987	RNASEL	0.617089
44	3123	AKT3	0.618574
				45	3687	CAMK4	0.61913
46	3303	CDKN2D	0.625741
				47	2966	NM_033266	0.627321
48	3226	RBX1	0.632166

49	3509	KIF21A	0.634426
				50	2999	FES	0.634596
51	3517	MYC	0.637624
				52	3592	SOS2	0.640343
53	3139	XM_095827	0.642535
				54	3105	BUB1	0.643861
55	3397	BUB3	0.644077
				56	3267	CCNH	0.64503
57	2975	NEK4	0.645485
				58	3766	S100A2	0.646739
59	2936	SGKL	0.64695
				60	3524	NOTCH3	0.647109
61	3806	C10orf3	0.64878
				62	3448	NM_016231	0.654135
63	3461	KIT	0.662033
				64	3501	RPS6KA2	0.667039
65	3494	MAPK4	0.668898
				66	3251	ABL1	0.675159
67	3103	PIK3CA	0.679543
				68	3572	RASA3	0.681105
69	3246	RPS6KB1	0.681548
				70	3230	MAP2K1	0.683985
71	3733	MYLK2	0.684534
				72	3491	PRKCE	0.685882
73	2982	CDC2L2	0.687831
				74	3542	PPP2CA	0.690237
75	3350	ADA	0.692046
				76	3651	VRK1	0.692308
77	2937	NM_025052	0.693089
				78	3007	MAP3K14	0.694169
79	3751	BCR	0.694278
				80	3410	RPS27	0.695238
81	3240	MAPK12	0.696498
				82	2949	MYO3B	0.698039
83	3413	KIF23	0.701413
				84	3773	WNT2	0.702997
85	3762	MCC	0.706533
				86	3731	CSNK1E	0.707275
87	3778	VHL	0.707386
				88	3476	PRKCD	0.708251
89	3754	CDKL2	0.712665
				90	3741	RPS6KB2	0.715261
91	3744	PRKWNK4	0.716089
				92	3148	LIG1	0.71816
93	2964	RIPK2	0.71873
				94	3486	RPS6KA3	0.71875
95	3772	RPS6KB1	0.722315
				96	3193	FGF3	0.723179
97	3363	GART	0.723732
				98	3438	DTR	0.725061
99	3351	ESR1	0.725395
				100	3416	IKBKE	0.726044

101	2972	KSR	0.727171
				102	3326	CCT7	0.727769
103	3648	CLK1	0.728232
				104	3401	HDAC1	0.728268
105	3498	CSNK1A1	0.728714
				106	2976	NEK7	0.729805
107	3347	top1	0.731826
				108	3236	PTK2B	0.736339
109	3256	CDC2L1	0.738272
				110	3606	CREBBP	0.738487
111	3657	PCTK1	0.739866
				112	3452	JAK1	0.745847
113	3250	CHEK2	0.745919
				114	3200	REL	0.746919
115	3403	AURKC	0.747841
				116	3663	ALS2CR2	0.749671
117	3208	ZW10	0.75
				118	3647	YES1	0.750637
119	3466	JAK3	0.750708
				120	3196	ARH1	0.75402
121	3757	CLK4	0.757793
				122	3434	DDX6	0.758671
123	3460	CSK	0.759454
				124	3722	TLK2	0.761568
125	3306	CDC14A	0.761859
				126	3412	KIF25	0.761959
127	2926	AF172264	0.762856
				128	3382	TUBB	0.763294
129	2965	NM_014720	0.763727
				130	3625	CTBP2	0.763827
131	3702	MAP3K13	0.764125
				132	3650	NM_025195	0.764957
133	3323	CDK5R1	0.765293
				134	3653	NPR2	0.765609
135	2997	MST1R	0.767068
				136	3658	STK18	0.768411
137	3739	NM_017886	0.768662
				138	2993	SRMS	0.768678
139	3166	PMS1	0.769717
				140	3775	NOTCH1	0.770983
141	3469	AAK1	0.772082
				142	3833	ATR	0.772423
143	3211	BUB1B	0.773389
				144	3557	JUND	0.773496
145	3179	PDGFB	0.777522
				146	3674	CSNK1D	0.779923
147	3566	JUN	0.780371
				148	3341	APLP2	0.781888
149	3188	PMS2	0.785359
				150	3633	CTBP1	0.786631
151	2923	ERN1	0.787194
				152	3086	KIF11	0.787201

153	3688	GUCY2D	0.787284
				154	3605	FZD4	0.787879
155	3640	STAT5B	0.789018
				156	2974	NEK11	0.791024
157	3473	DAPK1	0.791285
				158	3376	AGA	0.791586
159	3263	CDC2	0.792593
				160	3475	EPHB4	0.797463
161	3346	CCNK	0.79871
				162	3298	CCNE1	0.800418
163	3359	TUBA1	0.801205
				164	3609	FZD3	0.806613
165	3201	RARA	0.808157
				166	3394	HDAC6	0.810106
167	3770	TGFBR2	0.810897
				168	3258	MOS	0.811566
169	3541	PKD2	0.811594
				170	3822	GTSE1	0.814495
171	3450	MAP3K2	0.81592
				172	3577	RAB2L	0.816
173	3203	ITGA5	0.817391
				174	3838	PRKAA2	0.821543
175	3085	KIF5C	0.82316
				176	3477	FGFR4	0.824427
177	3573	NM_016848	0.824468
				178	3836	TP53	0.825022
179	3782	SOS1	0.825161
				180	3366	TYMS	0.828914
181	3381	POLR2B	0.828921
				182	3710	GPRK2L	0.830756
183	2934	IRAK2	0.830809
				184	3364	HPRT1	0.831103
185	3182	MYCN	0.831349
				186	3783	KRAS2	0.831863
187	3113	CNK	0.834672
				188	3835	CHEK2	0.836402
189	3680	CLK3	0.836728
				190	3131	KIF1B	0.83697
191	3088	KIF13B	0.838299
				192	3581	RASGRP1	0.839735
193	3829	KIF2C	0.840215
				194	3380	TUBG2	0.840866
195	3334	CUL4B	0.842773
				196	3746	HUNK	0.84279
197	2921	RPS6KA5	0.845122
				198	3769	PLAU	0.845466
199	2984	EPHB6	0.847067
				200	3814	HMMR	0.850166
201	3623	CTNND1	0.850309
				202	3444	NEK3	0.851852
203	2935	MAPK6	0.852713
				204	2996	MAPK3	0.853188

205	2969	NM_014916	0.856081
				206	3120	PIK3CB	0.856195
207	3107	KIF2	0.856252
				208	3502	PRKCH	0.856893
209	3763	NM_016231	0.858333
				210	3419	RFC4	0.858657
211	3639	STAT6	0.858685
				212	2930	ITK	0.860156
213	3124	KIF4A	0.860439
				214	3209	MAD2L2	0.860811
215	3832	ATM	0.861555
				216	3774	CDC45L	0.862319
217	3342	CCNT1	0.86272
				218	3430	STMN1	0.864508
219	3802	NOTCH3	0.865116
				220	3309	CCND2	0.865741
221	3411	HIF1A	0.867769
				222	3717	NTRK2	0.867864
223	3465	EPHA1	0.867876
				224	3795	NR4A2	0.867991
225	3659	NM_015978	0.868205
				226	3643	DDR2	0.868618
227	3392	BIRC5	0.869293
				228	3786	FRAP1	0.870607
229	3297	CCT2	0.872024
				230	2991	NPR1	0.872727
231	3318	CUL2	0.87438
				232	3293	CDC25A	0.875
233	3421	ORC6L	0.875341
				234	3454	FLT4	0.875663
235	2950	NEK6	0.876961
				236	3815	MAPRE2	0.877732
237	3831	CLSPN	0.878064
				238	3232	KDR	0.878378
239	3709	X95425	0.879358
				240	2929	CHUK	0.881491
241	3378	NM_006009	0.882129
				242	2952	PRKG1	0.883408
243	3776	NOTCH2	0.88366
				244	3356	TUBG1	0.884709
245	3308	CDKN2B	0.885077
				246	2967	NM_016653	0.886094
247	3591	RALB	0.889362
				248	3635	STAT1	0.889881
249	3530	NOTCH1	0.890111
				250	3750	CAMK2A	0.891525
251	3523	NOTCH2	0.893064
				252	2980	RIPK1	0.894417
253	3249	MAP2K6	0.895216
				254	3589	SOS1	0.895558
255	3587	VAV3	0.896552
				256	2968	STK17B	0.899438

257	3505	STK6	0.899549
				258	3526	HES6	0.899892
259	3261	ERBB2	0.904662
				260	3252	NEK2	0.904873
261	3426	TK1	0.906569
				262	3328	CCNC	0.909091
263	3470	RPS6KA1	0.909627
				264	3798	ACTR2	0.910468
265	3595	FEN1	0.910569
				266	3597	SHMT2	0.911368
267	3362	NR3C1	0.911404
				268	3257	IGF1R	0.911442
269	3665	PAK4	0.913313
				270	3678	PFTK1	0.913673
271	3344	CDKL1	0.913907
				272	3302	CDK8	0.913988
273	3536	PIK3C3	0.916089
				274	3685	LTK	0.917246
275	3749	NM_139021	0.917681
				276	3268	CDC25C	0.919654
277	3743	RAGE	0.922602
				278	3414	HDAC7A	0.925495
279	3162	MADH2	0.927277
				280	3429	MCM6	0.928214
281	3682	DYRK1A	0.928261
				282	3585	GAB1	0.928513
283	3549	PIK3R2	0.930054
				284	3233	ROCK1	0.930818
285	3315	CCT4	0.931751
				286	2990	NM_015112	0.933149
287	3409	TTK	0.934641
				288	3237	CDC7	0.938429
289	2960	LYN	0.938849
				290	3664	STK17A	0.93923
291	2931	MAP4K3	0.939649
				292	3693	NEK9	0.939894
293	3694	STK38	0.941537
				294	3000	BMX	0.94164
295	3445	BMPR1A	0.944154
				296	3207	MAD1L1	0.945191
297	3714	NM_013355	0.947122
				298	3652	PDGFRA	0.947533
299	3631	WISP2	0.948783
				300	3799	ACTR3	0.949315
301	2912	MPHOSPH1	0.95053
				302	3142	KIF25	0.950655
303	3755	CDKL3	0.950839
				304	3231	ILK	0.951
305	3155	ATR	0.951613
				306	3646	NM_005781	0.952566
307	2979	PAK2	0.953323
				308	3296	CDKN2C	0.954784

309	2983	GUCY2C	0.956701
				310	3497	EPHA8	0.958146
311	3163	THRA	0.959354
				312	3471	MAPK10	0.960665
313	2940	DCAMKL1	0.963487
				314	3593	VAV2	0.963865
315	3398	HDAC11	0.964784
				316	3752	CCNB3	0.964824
317	3641	TYRO3	0.965291
				318	3195	CDH1	0.96542
319	3552	PDK1	0.96695
				320	3132	KIF23	0.970496
321	2951	AJ311798	0.971591
				322	3214	CENPF	0.974453
323	3436	BRAF	0.97619
				324	3104	CDK4	0.976337
325	2959	PIM2	0.977075
				326	3228	CDC27	0.978723
327	3570	RALGDS	0.979927
				328	3826	NM_015694	0.981405
329	3788	PRKCE	0.983254
				330	2954	ROCK2	0.983541
331	3539	PLCG2	0.985447
				332	3732	CSNK2A2	0.985667
333	3759	NM_006622	0.98881
				334	2998	MAPK7	0.993015
335	3352	NR3C2	0.996058
				336	3488	AURKB	0.996508
337	3130	FRAP1	0.996898
				338	3691	NM_024046	0.997251
339	3683	NM_003138	0.998179
				340	3569	RASGRP2	1.000543
341	2920	EIF2AK3	1.005703
				342	3365	PRIM1	1.006112
343	3462	T6FBR2	1.00726
				344	3513	ROS1	1.009016
345	3102	CKS2	1.013052
				346	2945	CDC42BPB	1.01398
347	3656	PRKCN	1.016229
				348	3726	MAPKAPK2	1.016458
349	3002	CRKL	1.01909
				350	3670	MAP3K10	1.01919
351	3767	FZD3	1.019811
				352	3645	CASK	1.020319
353	3707	TXK	1.022666
				354	3455	MAP2K4	1.023218
355	3372	TUBA8	1.025814
				356	3540	PPP2CB	1.027826
357	3690	PRKAA2	1.029902
				358	3307	CDC6	1.03012
359	3495	FGFR2	1.032093
				360	3485	DHX8	1.032492

361	3696	NM_016281	1.038149
				362	3716	ULK1	1.039167
363	3265	RAF1	1.039442
				364	3161	WT1	1.039655
365	3215	MAD2L1	1.039783
				366	3415	HSPCA	1.040186
367	3127	MAPK1	1.040277
				368	3686	MAP3K8	1.040303
369	3490	ERBB3	1.040323
				370	3441	PRKCI	1.042373
371	3115	MDM2	1.04276
				372	3264	CDK3	1.044285
373	3147	CDKN2A	1.045872
				374	3568	PLD1	1.048696
375	3559	RAP1GDS1	1.05
				376	2928	IRAK1	1.050577
377	3197	ARHB	1.052064
				378	3785	GRB2	1.052525
379	3248	JAK2	1.053539
				380	3199	NF2	1.053654
381	2992	PRKR	1.055468
				382	3516	ARHA	1.058051
383	3449	TBK1	1.059537
				384	2953	MAPK11	1.059656
385	3164	MYCL1	1.060646
				386	3745	CAMKK2	1.061685
387	3324	CUL5	1.062571
				388	3243	NM_004203	1.062998
389	3187	WNT7B	1.063935
				390	3459	EGFR	1.066553
391	3239	CDK6	1.067257
				392	3170	BLM	1.068402
393	2943	DYRK2	1.06862
				394	3320	CCND3	1.070018
395	3369	NM_007027	1.071887
				396	3624	CTNNB1	1.072588
397	3500	SGK	1.074011
				398	3101	MAPK14	1.074871
399	3408	PIN1	1.075614
				400	2924	STK25	1.076046
401	3548	RAC1	1.07851
				402	3676	MAP4K1	1.079121
403	3698	ADRBK2	1.079569
				404	3301	CCNB1	1.080243
405	2925	FYN	1.081081
				406	3565	RAB2	1.081968
407	2977	R1PK3	1.082037
				408	3810	AI278633	1.084388
409	3796	ARHGEF6	1.084848
				410	3116	KIF5A	1.086755
411	3590	ARHGEF2	1.088083
				412	3679	CLK2	1.088737

413	3119	CDKN1B	1.089067
				414	3367	DHFR	1.092319
415	3797	ARHGEF9	1.092391
				416	3405	HDAC8	1.096856
417	2957	TYK2	1.099156
				418	3091	KIFC3	1.10008
419	3546	INPP5D	1.102828
				420	3227	NUMA1	1.104478
421	3181	ST5	1.104782
				422	3807	SPAG5	1.105317
423	3090	KIF3C	1.10597
				424	3343	CENPJ	1.107383
425	3245	AXL	1.108766
				426	3097	KIF20A	1.108842
427	3360	RRM2	1.109827
				428	3349	IMPDH1	1.111043
429	3474	CSNK2A1	1.111842
				430	3616	FZD1	1.11295
431	3620	AXIN2	1.113386
				432	2995	PTK2	1.115385
433	3634	BTRC	1.117674
				434	3504	PIK3CB	1.118194
435	3561	FOS	1.118649
				436	3618	DVL2	1.12
437	3537	EIF4EBP1	1.121316
				438	3550	PLCG1	1.121971
439	3443	EPS8	1.122744
				440	3370	AR	1.123767
441	3543	PDK2	1.12548
				442	3122	ATSV	1.127371
443	3167	S100A2	1.127907
				444	3596	SHMT1	1.128114
445	3811	NM_152524	1.129555
				446	3779	CTNNA1	1.129565
447	3312	CUL3	1.133047
				448	2963	MAP3K11	1.133758
449	2942	TTN	1.133889
				450	3790	ERBB3	1.135274
451	3094	KIF3A	1.13729
				452	3545	IRS2	1.139283
453	3305	CDC2L5	1.140753
				454	3748	NM_016507	1.140961
455	3614	CTNND2	1.141748
				456	3437	FGFR1	1.143284
457	3389	NM_052963	1.145845
				458	3213	NM_016238	1.145939
459	3533	HES7	1.148773
				460	3321	CDKN3	1.152745
461	3711	LIMK1	1.153559
				462	3503	EGR1	1.156344
463	3701	STK10	1.160598
				464	3608	MAP3K7IP1	1.161191

465	3730	TESK1	1.162946
				466	3156	MSH2	1.163507
467	3571	VAV1	1.164063
				468	3668	DAPK3	1.165365
469	3677	HCK	1.166105
				470	3708	RPS6KC1	1.166667
471	3110	KIF13A	1.167294
				472	3185	VCAM1	1.170254
473	3837	PRKAA1	1.171443
				474	3514	HRAS	1.171476
475	3371	NM_006087	1.175311
				476	3420	NM_014109	1.176378
477	3669	NTRK3	1.17801
				478	2939	TLK1	1.179137
479	3654	VRK2	1.180868
				480	3636	STAT2	1.181562
481	3506	XM_095827	1.18376
				482	3728	TIE	1.184901
483	3496	EIF4EBP2	1.188138
				484	2994	MATK	1.188439
485	3353	PGR	1.188925
				486	3771	PIK3CA	1.191131
487	3111	KIF5B	1.191167
				488	3396	HDAC10	1.192015
489	3330	CDK9	1.194303
				490	3705	NM_012119	1.195395
491	3339	CCNB2	1.195402
				492	3005	MERTK	1.196303
493	3220	ANAPC11	1.1994
				494	3507	NM_145754	1.199485
495	3418	CENPA	1.199564
				496	3492	PRKCQ	1.199597
497	3499	GRB2	1.204124
				498	3667	NM_016542	1.204923
499	3084	KIF14	1.207333
				500	3317	CCNI	1.208734
501	3457	BAD	1.2C8929
				502	3819	TACC3	1.209677
503	3377	NM_006088	1.210588
				504	3472	MAP3K5	1.210677
505	2922	NM_004783	1.211356
				506	3453	MAP2K2	1.212321
507	3724	EPHA7	1.213738
				508	3260	STK11	1.214815
509	3675	ADRBK1	1.215503
				510	3379	NM_032525	1.223512
511	2956	PRKCL2	1.223938
				512	3666	PAK6	1.229403
513	3216	CDC20	1.231173
				514	3672	SYK	1.231714
515	3555	RASA1	1.236402
				516	3354	RRM1	1.237695

517	3153	RB1	1.237825
				518	3253	PRKCA	1.239404
519	3146	KIF21A	1.240245
				520	3756	CDK5RAP2	1.242775
521	3721	ANKRD3	1.245185
				522	3224	FBXO5	1.24973
523	3607	TLE1	1.250329
				524	2981	ALK	1.252514
525	2978	AB067470	1.252713
				526	3440	FGFR3	1.253731
527	3578	RREB1	1.256567
				528	3393	HDAC2	1.258824
529	3520	NRAS	1.263715
				530	3190	WNT4	1.265328
531	3463	TEC	1.265973
				532	3621	CTNNA2	1.26658
533	3425	DLG7	1.267399
				534	3311	CDK10	1.269347
535	3567	SHC1	1.270057
				536	3753	CDK5	1.276163
537	2989	ACVR1B	1.276215
				538	3692	AB007941	1.27931
539	3244	PRKCZ	1.279368
				540	3092	KIF12	1.279896
541	3487	MAP2K3	1.280835
				542	3813	ANLN	1.282313
543	3198	ICAM1	1.285429
				544	3697	CAMK2G	1.286036
545	3735	PRKACB	1.286694
				546	3100	GSK3B	1.289078
547	3431	NM_018454	1.289806
				548	3615	FZD2	1.292222
549	2947	NM_007064	1.29381
				550	3340	CENPH	1.293935
551	3172	PLAU	1.297571
				552	3160	TACSTD1	1.297585
553	3212	NM_022662	1.301215
				554	3098	CENPE	1.305802
555	3626	DVL3	1.306682
				556	3830	NM_013296	1.307494
557	3713	PRKG2	1.307933
				558	3768	ARAF1	1.308011
559	3493	MAPK9	1.308449
				560	3108	K1F22	1.308726
561	3169	NME1	1.310985
				562	3125	NM_031217	1.311267
563	3375	AHCY	1.311852
				564	3583	JUNB	1.31241
565	3458	ARAF1	1.315519
				566	3612	TCF1	1.316285
567	3294	CCNF	1.317748
				568	3338	CUL4A	1.318527

569	3649	CAMK2B	1.322337
				570	3576	GRAP	1.322985
571	3527	DTX2	1.33023
				572	3145	C20orf23	1.334687
573	3180	CD44	1.335574
				574	3758	RAD51L1	1.335901
575	3165	FGF2	1.336082
				576	3828	KIF20A	1.337004
577	3553	CKS1B	1.339383
				578	3089	KIFCI	1.341566
579	3442	ERBB4	1.345118
				580	3554	PIK3R3	1.347147
581	3613	DVL1	1.347505
				582	2985	MKNK1	1.347934
583	3117	ATM	1.348967
				584	3424	EZH2	1.352941
585	3695	PRKAA1	1.355145
				586	3446	PRKCG	1.355556
587	3194	RARB	1.359932
				588	3644	EPHB1	1.36061
589	3700	AB037782	1.361005
				590	3599	DTYMK	1.361789
591	3729	RYK	1.361997
				592	3114	PIK3CD	1.362808
593	3821	ASPM	1.363705
				594	3373	top2A	1.363708
595	3563	RAB3A	1.365615
				596	3764	NOTCH4	1.36911
597	3628	CTNNBL1	1.3702
				598	3823	NM_017779	1.372126
599	3715	CDC42BPA	1.372256
				600	3562	RASD1	1.372563
601	3784	MAPK8	1.376577
				602	3574	SH3KBP1	1.384674
603	3594	RAP2A	1.393939
				604	3662	LCK	1.3981
605	3787	FZD4	1.399749
				606	3316	CCNA1	1.404295
607	3684	STK38L	1.406161
				608	3610	LEF1	1.407463
609	3390	NM_080925	1.407563
				610	3152	APC	1.414678
611	3149	TP53	1.420044
				612	3238	MAP3K3	1.420428
613	3109	KIF1C	1.420608
				614	3325	CDKN1C	1.42522
615	3314	CCNG1	1.426516
				616	3825	NM_152562	1.428805
617	3588	RHEB	1.435039
				618	3736	PTK7	1.440171
619	3118	NM_032559	1.440252
				620	3521	MET	1.440418

621	3096	AKT1	1.440951
				622	3361	IMPDH2	1.442308
623	3582	GRAP2	1.444349
				624	3584	RASAL2	1.450119
625	3801	PSEN1	1.466292
				626	3803	MPHOSPH1	1.470276
627	2938	ALS2CR7	1.471357
				628	3106	KIF9	1.47493
629	3313	CCNG2	1.48267
				630	3792	ARHGEF1	1.48329
631	3210	NM_013366	1.48366
				632	3820	NM_018410	1.483709
633	2932	MAPKAPK3	1.488
				634	3747	GSK3A	1.491773
635	2962	MAP4K2	1.495448
				636	3699	NM_032844	1.502812
637	3189	MYB	1.504618
				638	3629	AXIN1	1.505556
639	2941	DYRK3	1.505717
				640	3818	A1338451	1.511194
641	2919	OSR1	1.512906
				642	3140	XM_089006	1.518548
643	3229	PRKCL1	1.525203
				644	3510	CDK4	1.529837
645	3319	CCND1	1.531034
				646	3159	RET	1.536506
647	3242	PRKCB1	1.540024
				648	3519	NTRK1	1.547773
649	3808	CKAP2	1.554545
				650	2988	FRK	1.557214
651	2944	MARK1	1.557763
				652	2971	DAPK2	1.55938
653	3299	CUL1	1.560841
				654	3660	DMPK	1.5625
655	3515	PDGFRB	1.562977
				656	3522	KRAS2	1.564353
657	3004	MAP3K1	1.570175
				658	3395	HDAC5	1.571159
659	3468	ABL2	1.571225
				660	3529	DTX1	1.57276
661	3329	CDC42	1.580386
				662	3704	ACVR2B	1.58046
663	3827	NM_018123	1.581315
				664	3456	FLT1	1.583826
665	3310	CDC34	1.585818
				666	3331	CDC25B	1.585938
667	3368	top3A	1.58728
				668	3126	KIF3B	1.588728
669	3780	MCM3	1.590296
				670	3128	AKT2	1.592696
671	3598	PCNA	1.59319
				672	3535	SKP2	1.593333

673	2955	PAK1	1.59552
				674	3234	CDK2	1.596033
675	3138	KIF17	1.604846
				676	3632	WISP1	1.607319
677	3611	CTNNAL1	1.611386
				678	3300	CDC14B	1.611486
679	3511	XM_168069	1.614698
				680	3144	KIF4B	1.619674
681	3627	CTNNA1	1.620915
				682	3337	CDKN1A	1.626582
683	3202	MCC	1.627957
				684	3143	NM_017596	1.628521
685	3186	ETS1	1.635593
				686	3432	PRC1	1.637647
687	3556	RAP1A	1.638173
				688	3335	CDK5R2	1.656172
689	2933	MAP4K5	1.656522
				690	2927	MAPK13	1.659401
691	2973	NEK1	1.664311
				692	3538	NFKB2	1.667808
693	3602	MCM3	1.678819
				694	3603	POLS	1.678937
695	3630	WISP3	1.679045
				696	3447	PRKCM	1.680152
697	3402	HDAC4	1.68123
				698	3133	XM_168069	1.681935
699	3428	ECT2	1.690096
				700	3720	AB002301	1.691718
701	3793	MAPRE1	1.693966
				702	3681	SRPK1	1.700611
703	3817	NM_019013	1.702326
				704	3136	XM_066649	1.708388
705	3355	GUK1	1.710938
				706	3087	PTEN	1.716866
707	3579	PDZGEF2	1.717714
				708	3168	DCC	1.719083
709	3151	MLH1	1.72077
				710	3217	NM_014885	1.722045
711	3191	WNT2	1.728016
				712	3765	CREBBP	1.72973
713	3655	CAMK2D	1.733773
				714	3407	HDAC9	1.748784
715	3255	CDK7	1.75
				716	3295	CDK2AP1	1.75
717	3192	WNT1	1.751208
				718	3333	CCNT2	1.761104
719	3703	AK024504	1.764425
				720	3760	CDKL5	1.769444
721	2948	MYO3A	1.769759
				722	3800	CHFR	1.772809
723	3544	IRS1	1.776668
				724	3235	CHEK1	1.776886

725	3137	KIF26A	1.782366
				726	3673	DDR1	1.792507
727	3336	CDC37	1.807985
				728	3725	EPHA4	1.820076
729	3404	PPARG	1.822581
				730	3604	TK2	1.82846
731	3738	PRKWNK3	1.836245
				732	3141	NM_145754	1.843889
733	3451	MAP3K4	1.855556
				734	3417	HDAC3	1.857143
735	3508	KIF25	1.871592
				736	3575	LATS2	1.879574
737	3761	WT1	1.88089
				738	3723	NM_018401	1.88722
739	3719	BMPR2	1.890545
				740	3204	CDC16	1.892826
741	3467	MAP2K7	1.894459
				742	2986	ACVR2	1.896882
743	3218	CDC23	1.904255
				744	3791	NM_005200	1.913043
745	3804	NM_024322	1.920139
				746	3558	RALA	1.92029
747	3824	MAPRE3	1.940871
				748	3622	FZD9	1.988166
749	3205	NM_139286	1.997054
				750	3221	top3B	1.997534
751	3794	WASL	1.998403
				752	3637	STAT4	2.005199
753	3834	CHEK1	2.01625
				754	3400	BCL2	2.045028
755	3223	NM_016263	2.045139
				756	3358	top2B	2.050562
757	3512	TGFBR1	2.062016
				758	3259	MAPK8	2.064081
759	3742	RHOK	2.075949
				760	2946	NM_017719	2.078131
761	3406	TERT	2.10274
				762	3206	ANAPC5	2.159615
763	3531	NM_021170	2.163086
				764	3008	SGK2	2.1766
765	3706	C20orf97	2.1875
				766	3254	CSF1R	2.196822
767	3439	EGR2	2.213333
				768	2970	AATK	2.235211
769	3528	TCF3	2.273649
				770	3327	CDC45L	2.288265
771	3551	STAT3	2.29125
				772	3001	PRKY	2.313131
773	3734	BMPR1B	2.330839
				774	3095	KIF2C	2.336785
775	3222	PTTGI	2.347826
				776	3532	NM_019089	2.352437

777	3547	FOXO1A	2.352444
				778	3671	STK4	2.362408
779	3781	SRC	2.37859
				780	3789	ELKI	2.394828
781	3247	NM_018492	2.480851
				782	3586	RASA2	2.506796
783	3727	GPRK6	2.553987
				784	3689	BLK	2.584588
785	3777	ABL1	2.615226
				786	3399	HSPCB	2.632207
787	2958	PRKACA	2.635514
				788	3304	CCNE2	2.677656
789	3617	CTNNBIP1	2.698292
				790	3225	NM_013367	2.714286
791	3619	FRAT1	2.728111
				792	3121	PIK3C2A	2.828125
793	3816	NM_017769	2.847273
				794	3134	XM_170783	2.923286
795	3737	NM_016457	2.940451
				796	3135	XM_064050	3.063002
797	3129	STK6	3.146434
				798	3564	RALBP1	3.170605
799	3580	ELK1	3.356401
				800	3157	NF1	3.402273
801	3638	STAT5A	3.754386
				802	3241	WEE1	3.801887
803	3718	PTK6	4.317857
				804	3712	RPS6KA6	5.356624
805	3158	BRCA1	5.821429
				806	3642	EPHB3	6.43
807	3150	BRCA2	14.13136

The average sensitization multiple that Table II C Zorubicin causes

Gene I	BiolD	Gene	The mean value of three screenings
				1	2514	PLK	0.094489
2	3099	PLK	0.195626
				3	3099	PLK	0.211482
4	3099	PLK	0.211747
				5	3099	PLK	0.219626
6	3099	PLK	0.227603
				7	3099	PLK	0.235482
8	3099	PLK	0.235683
				9	3099	PLK	0.235747
10	3099	PLK	0.251539
				11	3099	PLK	0.251603
12	3099	PLK	0.259683
				13	3099	PLK	0.275539
14	3099	PLK	0.282503

15	3099	PLK	0.298359
				16	3099	PLK	0.298624
17	3099	PLK	0.31448
				18	3099	PLK	0.32256
19	3534	PLK	0.330807
				20	3099	PLK	0.338416
21	3099	PLK	0.395491
				22	3099	PLK	0.411612
23	3006	PLK	0.415454
				24	3099	PLK	0.419491
25	3099	PLK	0.435548
				26	3099	PLK	0.435612
27	3433	PLK	0.435845
				28	3391	PLK	0.440842
29	3099	PLK	0.459548
				30	3099	PLK	0.482368
31	3099	PLK	0.498489
				32	3322	CCNA2	0.512614
33	3099	PLK	0.522425
				34	3805	C20orf1	0.562328
35	3423		0.613084
				36	3600	RRM2B	0.659243
37	3305	CDC2L5	0.68014
				38	3542	PPP2CA	0.695506
39	3266	PLK	0.696721
				40	3228	CDC27	0.70157
41	3464	INSR	0.70706
				42	3326	CCT7	0.724986
43	3740	STK35	0.754807
				44	3731	CSNK1E	0.765738
45	3416	IKBKE	0.773235
				46	3293	CDC25A	0.77957
47	3309	CCND2	0.791487
				48	3350	ADA	0.800034
49	3812	CDCA8	0.815766
				50	3354	RRM1	0.817751
51	3446	PRKCG	0.822809
				52	3648	CLK1	0.824307
53	3509	KIF21A	0.826427
				54	3526	HES6	0.826991
55	3250	CHEK2	0.828202
				56	3262	LATS1	0.82944
57	3359	TUBA1	0.839308
				58	3344	CDKL1	0.840425
59	2984	EPHB6	0.846685
				60	3702	MAP3K13	0.84685
61	3838	PRKAA2	0.853115
				62	3422	SMC4L1	0.854651
63	3332	CDC5L	0.85491
				64	3750	CAMK2A	0.857171
65	3686	MAP3K8	0.8599
				66	3226	RBX1	0.862335

67	3438	DTR	0.863218
				68	3318	CUL2	0.863485
69	3454	FLT4	0.864511
				70	3366	TYMS	0.866092
71	3444	NEK3	0.866318
				72	3397	BUB3	0.867363
73	3007	MAP3K14	0.86906
				74	3373	top2A	0.875387
75	2934	IRAK2	0.875671
				76	3188	PMS2	0.876644
77	3461	KIT	0.876727
				78	3398	HDAC11	0.878587
79	3665	PAK4	0.879213
				80	3494	MAPK4	0.879947
81	3303	CDKN2D	0.88429
				82	2925	FYN	0.885569
83	3437	FGFR1	0.889075
				84	3219	CENPC1	0.889832
85	3491	PRKCE	0.891708
				86	3105	BUB1	0.892262
87	3609	FZD3	0.89297
				88	3421	ORC6L	0.893859
89	3414	HDAC7A	0.894925
				90	3342	CCNT1	0.89645
91	3193	FGF3	0.897275
				92	3203	ITGA5	0.89915
93	3679	CLK2	0.899792
				94	3656	PRKCN	0.903305
95	3677	HCK	0.903727
				96	3172	PLAU	0.904045
97	2999	FES	0.904351
				98	3161	WT1	0.907863
99	3230	MAP2K1	0.908157
				100	2937		0.910875
101	3502	PRKCH	0.913184
				102	3317	CCN1	0.913695
103	3086	KIF11	0.914508
				104	3412	KIF25	0.915671
105	3710	GPRK2L	0.917359
				106	3585	GAB1	0.91762
107	3807	SPAG5	0.918025
				108	3815	MAPRE2	0.919461
109	3646		0.920311
				110	3000	BMX	0.920926
111	3365	PRIM1	0.922943
				112	3574	SH3KBP1	0.924261
113	3485	DHX8	0.924589
				114	3527	DTX2	0.92511
115	3378		0.927814
				116	3799	ACTR3	0.929286
117	3822	GTSE1	0.929871
				118	3100	GSK3B	0.932676

119	3206	ANAPC5	0.932816
				120	3351	ESR1	0.932858
121	3623	CTNND1	0.932974
				122	3601	POLE	0.935664
123	3097	KIF20A	0.939338
				124	2991	NPR1	0.941392
125	2926		0.943073
				126	3717	NTRK2	0.94323
127	3162	MADH2	0.953335
				128	3783	KRAS2	0.954957
129	3660	DMPK	0.955308
				130	3236	PTK2B	0.955874
131	3088	KIF13B	0.960206
				132	3774	CDC45L	0.961565
133	3540	PPP2CB	0.96255
				134	3251	ABL1	0.96267
135	3498	CSNK1A1	0.963185
				136	3307	CDC6	0.963749
137	3830		0.96419
				138	3374	POLR2A	0.964327
139	3413	KIF23	0.967774
				140	3296	CDKN2C	0.967818
141	3132	KIF23	0.96794
				142	3708	RPS6KC1	0.969675
143	3445	BMPR1A	0.970178
				144	3694	STK38	0.970842
145	3566	JUN	0.971389
				146	3140		0.97186
147	3571	VAV1	0.972374
				148	2993	SRMS	0.972957
149	3268	CDC25C	0.973198
				150	3835	CHEK2	0.973353
151	3557	JUND	0.973868
				152	3195	CDH1	0.973895
153	3375	AHCY	0.974215
				154	3163	THRA	0.976052
155	3164	MYCL1	0.979364
				156	3798	ACTR2	0.980521
157	3392	BIRC5	0.980792
				158	3196	ARHI	0.980973
159	3536	PIK3C3	0.981403
				160	2950	NEK6	0.981709
161	3773	WNT2	0.982648
				162	3776	NOTCH2	0.983584
163	3814	HMMR	0.983597
				164	3234	CDK2	0.983724
165	2982	CDC2L2	0.984121
				166	3826		0.985249
167	2953	MAPK11	0.987788
				168	3403	AURKC	0.988679
169	3586	RASA2	0.989648
				170	3503	EGR1	0.991443

171	3166	PMS1	0.99314
				172	3394	HDAC6	0.994139
173	3652	PDGFRA	0.994658
				174	3625	CTBP2	0.994928
175	3294	CCNF	0.995133
				176	3260	STK11	0.998488
177	2968	STK17B	0.998826
				178	3703		0.999818
179	3577	RAB2L	1.00021
				180	3184	TSG101	1.00109
181	2927	MAPK13	1.001159
				182	3116	KIF5A	1.002239
183	3496	EIF4EBP2	1.005451
				184	3741	RPS6KB2	1.00589
185	3298	CCNE1	1.005922
				186	2990		1.006496
187	3142	KIF25	1.006522
				188	3218	CDC23	1.009586
189	3517	MYC	1.010689
				190	2997	MST1R	1.011122
191	3003	FER	1.012506
				192	3700		1.013542
193	3470	RPS6KA1	1.013802
				194	3439	EGR2	1.013847
195	3429	MCM6	1.014653
				196	3372	TUBA8	1.017048
197	3556	RAP1A	1.017133
				198	3155	ATR	1.017435
199	3649	CAMK2B	1.017461
				200	3501	RPS6KA2	1.018616
201	3336	CDC37	1.019161
				202	2928	IRAK1	1.021732
203	3733	MYLK2	1.021742
				204	2960	LYN	1.022112
205	3301	CCNB1	1.022891
				206	3743	RAGE	1.023372
207	3525	NOTCH4	1.02341
				208	3767	FZD3	1.023646
209	2954	ROCK2	1.02397
				210	3475	EPHB4	1.024709
211	3635	STAT1	1.026128
				212	3746	HUNK	1.026176
213	2977	RIPK3	1.0272
				214	3573		1.028343
215	3751	BCR	1.028418
				216	3112	KNSL7	1.029109
217	3488	AURKB	1.029885
				218	3356	TUBG1	1.029908
219	3364	HPRT1	1.030247
				220	3465	EPHA1	1.032043
221	3828	KIF20A	1.032108
				222	3434	DDX6	1.03425

223	3143		1.03439
				224	3212		1.034473
225	3725	EPHA4	1.034871
				226	3473	DAPK1	1.035466
227	3581	RASGRP1	1.036407
				228	3357	PRIM2A	1.036773
229	3469	AAK1	1.037538
				230	3171	VHL	1.038422
231	3123	AKT3	1.039278
				232	3572	RASA3	1.04084
233	3615	FZD2	1.042378
				234	3658	STK18	1.043083
235	3261	ERBB2	1.044345
				236	3220	ANAPC11	1.0449
237	3639	STAT6	1.045395
				238	2959	PIM2	1.048207
239	2935	MAPK6	1.050943
				240	3752	CCNB3	1.051148
241	3431		1.05315
				242	3101	MAPK14	1.054104
243	3462	TGFBR2	1.056272
				244	3319	CCND1	1.057299
245	3592	SOS2	1.058842
				246	3655	CAMK2D	1.061571
247	3513	ROS1	1.062804
				248	3297	CCT2	1.064889
249	3549	PIK3R2	1.066314
				250	2998	MAPK7	1.066798
251	3334	CUL4B	1.066807
				252	3381	POLR2B	1.068615
253	3633	CTBP1	1.069269
				254	3678	PFTK1	1.07042
255	2987	RNASEL	1.072118
				256	3256	CDC2L1	1.073967
257	3558	RALA	1.074961
				258	3749		1.075156
259	3252	NEK2	1.075822
				260	2919	OSR1	1.077885
261	3393	HDAC2	1.077906
				262	3747	GSK3A	1.078401
263	3410	RPS27	1.078517
				264	3107	KIF2	1.078686
265	3654	VRK2	1.081195
				266	3533	HES7	1.081287
267	2983	GUCY2C	1.083605
				268	3555	RASA1	1.084083
269	3258	MOS	1.084874
				270	3180	CD44	1.085294
271	3124	KIF4A	1.086165
				272	3179	PDGFB	1.086599
273	3209	MAD2L2	1.088835
				274	3295	CDK2AP1	1.089703

275	3726	MAPKAPK2	1.09032
				276	3674	CSNK1D	1.090974
277	3616	FZD1	1.091935
				278	3452	JAK1	1.092015
279	3823		1.092187
				280	3745	CAMKK2	1.092307
281	3149	TP53	1.092755
				282	3561	FOS	1.092859
283	3836	TP53	1.093641
				284	3170	BLM	1.094952
285	2930	ITK	1.095322
				286	3744	PRKWNK4	1.095854
287	3401	HDAC1	1.096531
				288	3300	CDC14B	1.096651
289	3348	DCK	1.096689
				290	3405	HDAC8	1.096956
291	3239	CDK6	1.097696
				292	3640	STAT5B	1.098035
293	2992	PRKR	1.098133
				294	3548	RAC1	1.09835
295	3306	CDC14A	1.098874
				296	2943	DYRK2	1.099617
297	3127	MAPK1	1.102044
				298	3716	ULK1	1.104258
299	2922		1.106102
				300	3160	TACSTD1	1.107397
301	2964	RIPK2	1.109482
				302	3634	BTRC	1.110007
303	3576	GRAP	1.110227
				304	3833	ATR	1.110614
305	3837	PRKAA1	1.111073
				306	2939	TLK1	1.111429
307	3125		1.11143
				308	3299	CUL1	1.111864
309	3813	ANLN	1.112297
				310	3756	CDK5RAP2	1.112508
311	2976	NEK7	1.112602
				312	2965		1.11309
313	3784	MAPK8	1.114132
				314	3653	NPR2	1.115282
315	3302	CDK8	1.115429
				316	3628	CTNNBL1	1.115905
317	3664	STK17A	1.115938
				318	3504	PIK3CB	1.117357
319	3395	HDAC5	1.118952
				320	3369		1.119457
321	3243		1.119572
				322	3715	CDC42BPA	1.119975
323	2924	STK25	1.122952
				324	3568	PLD1	1.123878
325	3676	MAP4K1	1.124218
				326	3343	CENPJ	1.127564

327	3238	MAP3K3	1.127647
				328	3424	EZH2	1.127778
329	3418	CENPA	1.128399
				330	3829	KIF2C	1.128457
331	3476	PRKCD	1.128572
				332	3407	HDAC9	1.129023
333	2951		1.13065
				334	3685	LTK	1.130723
335	2942	TTN	1.131132
				336	3085	KIF5C	1.133235
337	3367	DHFR	1.133721
				338	3362	NR3C1	1.134725
339	3400	BCL2	1.134785
				340	3800	CHFR	1.134967
341	3103	PIK3CA	1.135082
				342	3711	LIMK1	1.135687
343	3165	FGF2	1.136323
				344	3213		1.137328
345	3370	AR	1.137843
				346	3772	RPS6KB1	1.138023
347	3189	MYB	1.138695
				348	3631	WISP2	1.138989
349	2945	CDC42BPB	1.140434
				350	3593	VAV2	1.141048
351	3338	CUL4A	1.141509
				352	3092	K1F12	1.14183
353	3782	SOS1	1.14272
				354	2989	ACVR1B	1.143948
355	3808	CKAP2	1.144074
				356	3310	CDC34	1.14429
357	3760	CDKL5	1.144621
				358	3159	RET	1.144761
359	3508	KIF25	1.144865
				360	3788	PRKCE	1.145993
361	3231	ILK	1.146387
				362	3471	MAPK10	1.146497
363	3668	DAPK3	1.14781
				364	3595	FEN1	1.14853
365	3775	NOTCH1	1.150372
				366	3145	C20orf23	1.151785
367	3570	RALGDS	1.152146
				368	2972	KSR	1.152379
369	3441	PRKCI	1.152901
				370	3737		1.153373
371	3463	TEC	1.154814
				372	3748		1.155977
373	3816		1.156751
				374	3582	GRAP2	1.158058
375	3360	RRM2	1.158514
				376	3516	ARHA	1.15962
377	3312	CUL3	1.160258
				378	3005	MERTK	1.160604

379	3456	FLT1	1.160651
				380	3567	SHC1	1.161312
381	3647	YES1	1.161861
				382	3447	PRKCM	1.162427
383	3739		1.163543
				384	3181	ST5	1.163581
385	3466	JAK3	1.164099
				386	3311	CDK10	1.1651
387	3486	RPS6KA3	1.165517
				388	3779	CTNNA1	1.165697
389	3148	LIG1	1.166358
				390	3683		1.167226
391	3544	IRS1	1.167527
				392	3335	CDK5R2	1.167989
393	3821	ASPM	1.167998
				394	3108	KIF22	1.168525
395	3168	DCC	1.170395
				396	3182	MYCN	1.172038
397	3119	CDKN1B	1.172505
				398	3692		1.173629
399	3687	CAMK4	1.17436
				400	3420		1.175153
401	3762	MCC	1.175576
				402	3519	NTRK1	1.175989
403	3257	IGF1R	1.176551
				404	3769	PLAU	1.176774
405	3339	CCNB2	1.177549
				406	3682	DYRK1A	1.178203
407	3240	MAPK12	1.178713
				408	3156	MSH2	1.17907
409	2936	SGKL	1.17989
				410	2920	EIF2AK3	1.179969
411	3670	MAP3K10	1.180357
				412	3207	MAD1L1	1.181963
413	3630	WISP3	1.182009
				414	3153	RB1	1.183084
415	3632	WISP1	1.183165
				416	3824	MAPRE3	1.183387
417	3624	CTNNB1	1.18419
				418	3151	MLH1	1.185254
419	3495	FGFR2	1.185537
				420	3349	IMPDH1	1.185827
421	2932	MAPKAPK3	1.186058
				422	3130	FRAP1	1.186158
423	3714		1.188036
				424	3467	MAP2K7	1.188179
425	3727	GPRK6	1.188457
				426	3500	SGK	1.189014
427	3638	STAT5A	1.189492
				428	3242	PRKCB1	1.191673
429	3588	RHEB	1.194214
				430	2940	DCAMKL1	1.194443

431	3222	PTTG1	1.194583
				432	3411	HIF1A	1.194933
433	2952	PRKG1	1.197336
				434	3539	PLCG2	1.198326
435	3797	ARHGEF9	1.20036
				436	2969		1.201116
437	3194	RARB	1.201145
				438	3490	ERBB3	1.202371
439	3197	ARHB	1.2033
				440	3347	top1	1.203483
441	2966		1.203678
				442	3089	KIFC1	1.204418
443	3232	KDR	1.205127
				444	3090	KIF3C	1.205488
445	3599	DTYMK	1.205645
				446	3139		1.206674
447	3695	PRKAA1	1.20878
				448	3425	DLG7	1.209098
449	3535	SKP2	1.20949
				450	3327	CDC45L	1.209854
451	3651	VRK1	1.210029
				452	3569	RASGRP2	1.210373
453	3246	RPS6KB1	1.210471
				454	3131	KIF1B	1.21146
455	3671	STK4	1.212033
				456	3757	CLK4	1.212447
457	2985	MKNK1	1.212627
				458	2988	FRK	1.213049
459	3432	PRC1	1.2136
				460	3699		1.214212
461	3008	SGK2	1.21451
				462	2996	MAPK3	1.217258
463	3399	HSPCB	1.217278
				464	3610	LEF1	1.219128
465	2980	RIPK1	1.220712
				466	3675	ADRBK1	1.22227
467	3663	ALS2CR2	1.223782
				468	3468	ABL2	1.223785
469	2961	PIM1	1.223874
				470	3804		1.224331
471	3594	RAP2A	1.226644
				472	3377		1.227759
473	3341	APLP2	1.229895
				474	3524	NOTCH3	1.230078
475	3253	PRKCA	1.233866
				476	3518	NFKB1	1.23456
477	3328	CCNC	1.236473
				478	3563	RAB3A	1.237081
479	3765	CREBBP	1.23722
				480	2979	PAK2	1.237809
481	3235	CHEK1	1.239472
				482	3146	KIF21A	1.239694

483	3340	CENPH	1.239979
				484	3215	MAD2L1	1.24524
485	3379		1.245359
				486	3662	LCK	1.245594
487	3754	CDKL2	1.247567
				488	3187	WNT7B	1.247786
489	3552	PDK1	1.248615
				490	3618	DVL2	1.249105
491	3602	MCM3	1.249136
				492	3564	RALBP1	1.249919
493	3404	PPARG	1.252208
				494	3248	JAK2	1.252557
495	3147	CDKN2A	1.252718
				496	3358	top2B	1.253573
497	3459	EGFR	1.255853
				498	3249	MAP2K6	1.256087
499	3254	CSF1R	1.258457
				500	2949	MYO3B	1.259934
501	3157	NF1	1.260606
				502	3680	CLK3	1.262403
503	3113	CNK	1.262742
				504	3825		1.263089
505	3667		1.26407
				506	3753	CDK5	1.264077
507	3553	CKS1B	1.265301
				508	2933	MAP4K5	1.2656S5
509	3796	ARHGEF6	1.265751
				510	3419	RFC4	1.266922
511	3460	CSK	1.266969
				512	3094	KIF3A	1.26728
513	3736	PTK7	1.267303
				514	3707	TXK	1.268516
515	3791		1.26868
				516	3523	NOTCH2	1.26965
517	3755	CDKL3	1.271644
				518	3204	CDC16	1.271646
519	3353	PGR	1.271733
				520	3115	MDM2	1.274517
521	3126	KIF3B	1.274522
				522	3095	KIF2C	1.274859
523	2947		1.277515
				524	3408	PIN1	1.278984
525	3657	PCTK1	1.279578
				526	3211	BUB1B	1.282741
527	3643	DDR2	1.28316
				528	3449	TBK1	1.285291
529	3669	NTRK3	1.285519
				530	3200	REL	1.285524
531	3729	RYK	1.291213
				532	3691		1.291243
533	3214	CENPF	1.291507
				534	3801	PSEN1	1.291634

535	2978		1.293122
				536	3141		1.294102
537	3792	ARHGEF1	1.294579
				538	3477	FGFR4	1.29633
539	3169	NME1	1.298008
				540	3693	NEK9	1.299989
541	3583	JUNB	1.300395
				542	3768	ARAF1	1.302137
543	2975	NEK4	1.302558
				544	3221	top3B	1.30285
545	3478	EPHA2	1.30329
				546	3666	PAK6	1.303653
547	2963	MAP3K11	1.306508
				548	3199	NF2	1.307337
549	3724	EPHA7	1.308035
				550	3457	BAD	1.308937
551	3185	VCAM1	1.309542
				552	3244	PRKCZ	1.310965
553	3587	VAV3	1.31294
				554	3712	RPS6KA6	1.313627
555	3216	CDC20	1.315701
				556	3551	STAT3	1.316414
557	3590	ARHGEF2	1.316547
				558	3659		1.317441
559	3831	CLSPN	1.318145
				560	3109	KIF1C	1.318847
561	3455	MAP2K4	1.319428
				562	3118		1.319892
563	3709		1.320996
				564	3122	ATSV	1.321439
565	3809	CDCA3	1.329173
				566	3237	CDC7	1.330145
567	3650		1.335065
				568	3382	TUBB	1.336093
569	3190	WNT4	1.336703
				570	3591	RALB	1.338466
571	3091	KIFC3	1.339318
				572	3761	WT1	1.340453
573	3832	ATM	1.343275
				574	3154	MADH4	1.343448
575	3002	CRKL	1.345404
				576	2946		1.346714
577	3389		1.347114
				578	3645	CASK	1.34749
579	3315	CCT4	1.348613
				580	3150	BRCA2	1.34964
581	3474	CSNK2A1	1.350654
				582	3458	ARAF1	1.351534
583	3528	TCF3	1.354214
				584	3529	DTX1	1.357117
585	3559	RAPIGDS1	1.359432
				586	3721	ANKRD3	1.361311

587	3819	?TACC3	1.367136
				588	3578	?RREB1	1.367845
589	3245	?AXL	1.367989
				590	3543	?PDK2	1.368231
591	3352	?NR3C2	1.368397
				592	3493	?MAPK9	1.368899
593	2958	?PRKACA	1.371294
				594	3435	?FLT3	1.371521
595	3316	?CCNA1	1.37217
				596	3263	?CDC2	1.373177
597	3224	?FBXO5	1.374389
				598	2701		1.378002
599	3497	?EPHA8	1.378119
				600	3255	?CDK7	1.379045
601	3766	?S100A2	1.379101
				602	3690	?PRKAA2	1.383537
603	3152	?APC	1.384859
				604	3201	?RARA	1.387549
605	3396	?HDAC10	1.391302
				606	3363	?GART	1.392568
607	2957	?TYK2	1.392639
				608	3323	?CDK5R1	1.394769
609	3380	?TUBG2	1.401159
				610	3233	?ROCK1	1.404806
611	3806	?C10orf3	1.405976
				612	3614	?CTNND2	1.409777
613	3093	?PIK3CG	1.41077
				614	3763		1.412316
615	3487	?MAP2K3	1.412348
				616	3732	?CSNK2A2	1.414035
617	3110	?KIF13A	1.414042
				618	3789	?ELK1	1.414448
619	3786	?FRAP1	1.416676
				620	3554	?PIK3R3	1.418107
621	3167	?S100A2	1.418532
				622	3084	?KIF14	1.41956
623	3661	?FGR	1.423887
				624	3617	?CTNNBIP1	1.425161
625	2974	?NEK11	1.426945
				626	3330	?CDK9	1.428872
627	3227	?NUMA1	1.432118
				628	3734	?BMPR1B	1.437299
629	3138	?KIF17	1.441566
				630	3186	?ETS1	1.442612
631	3673	?DDR1	1.444582
				632	3450	?MAP3K2	1.446117
633	3133		1.450561
				634	3598	?PCNA	1.451899
635	3106	?KIF9	1.45407
				636	3608	?MAP3K7IP1	1.455728
637	3376	?AGA	1.457466
				638	3443	?EPS8	1.460769

639	3102	CKS2	1.464872
				640	3409	TTK	1.465445
641	3346	CCNK	1.465948
				642	3604	TK2	1.466617
643	2921	RPS6KA5	1.467418
				644	3597	SHMT2	1.468236
645	3499	GRB2	1.469395
				646	3406	TERT	1.482496
647	3158	BRCA1	1.485158
				648	3114	PIK3CD	1.485825
649	3575	LATS2	1.487058
				650	3390		1.487135
651	3596	SHMT1	1.487573
				652	3514	HRAS	1.488051
653	3730	TESK1	1.489848
				654	3620	AXIN2	1.491167
655	3619	FRAT1	1.491691
				656	3644	EPHB1	1.492026
657	3117	ATM	1.498897
				658	3541	PKD2	1.5005
659	3607	TLE1	1.501123
				660	3229	PRKCL1	1.502059
661	3104	CDK4	1.502301
				662	3684	STK38L	1.5024
663	3626	DVL3	1.504253
				664	2986	ACVR2	1.510627
665	2971	DAPK2	1.516585
				666	3759		1.517994
667	3636	STAT2	1.519082
				668	3611	CTNNAL1	1.523973
669	3794	WASL	1.529001
				670	2944	MARK1	1.53037
671	3713	PRKG2	1.535337
				672	3087	PTEN	1.540121
673	3506		1.54045
				674	3191	WNT2	1.553178
675	3202	MCC	1.554866
				676	3210		1.555868
677	3738	PRKWNK3	1.559877
				678	3096	AKT1	1.560781
679	3308	CDKN2B	1.566367
				680	3606	CREBBP	1.570055
681	3641	TYRO3	1.574144
				682	3758	RAD51L1	1.575115
683	3192	WNT1	1.579448
				684	3705		1.591723
685	3565	RAB2	1.597556
				686	3770	TGFBR2	1.602887
687	3771	PIK3CA	1.605624
				688	3314	CCNG1	1.606354
689	3579	PDZGEF2	1.609017
				690	3603	POLS	1.609696

691	3589	SOS1	1.610658
				692	2938	ALS2CR7	1.613751
693	3621	CTNNA2	1.62379
				694	3265	RAF1	1.625904
695	3698	ADRBK2	1.626836
				696	3622	FZD9	1.630823
697	3111	KIF5B	1.633474
				698	3688	GUCY2D	1.63373
699	3489	SRC	1.633931
				700	3320	CCND3	1.636023
701	2970	AATK	1.636349
				702	3562	RASD1	1.636677
703	3728	TIE	1.637314
				704	3827		1.638302
705	3778	VHL	1.639913
				706	3448		1.6515
707	3426	TK1	1.654609
				708	2701		1.655539
709	3331	CDC25B	1.661891
				710	3371		1.664564
711	2923	ERN1	1.665407
				712	3550	PLCG1	1.667241
713	3803	MPHOSPH1	1.668632
				714	3333	CCNT2	1.669848
715	3520	NRAS	1.670763
				716	3121	PIK3C2A	1.675061
717	3264	CDK3	1.681459
				718	3785	GRB2	1.681539
719	3205		1.682938
				720	3811		1.685119
721	3507		1.688535
				722	2955	PAK1	1.688691
723	3440	FGFR3	1.695183
				724	2994	MATK	1.696094
72S	2967		1.703715
				726	3325	CDKN1C	1.703926
727	3545	IRS2	1.705996
				728	3492	PRKCQ	1.706638
729	3547	FOXO1A	1.710389
				730	3530	NOTCH1	1.711344
731	3810		1.723959
				732	3321	CDKN3	1.724044
733	3453	MAP2K2	1.737418
				734	3793	MAPRE1	1.738248
735	2941	DYRK3	1.741034
				736	3217		1.745349
737	3451	MAP3K4	1.753145
				738	3442	ERBB4	1.760041
739	3696		1.760172
				740	3701	STK10	1.767448
741	3817		1.768117
				742	2912	MPHOSPH1	1.771582

743	3001	PRKY	1.772697
				744	3128	AKT2	1.773864
745	2981	ALK	1.781796
				746	3337	CDKN1A	1.781903
747	3802	NOTCH3	1.787122
				748	3735	PRKACB	1.790032
749	3183		1.793085
				750	3430	STMN1	1.798292
751	3531		1.800094
				752	3515	PDGFRB	1.80459
753	3324	CUL5	1.820969
				754	3511		1.832738
755	3472	MAP3K5	1.834487
				756	3428	ECT2	1.84097
757	3642	EPHB3	1.84828
				758	3208	ZW10	1.858453
759	3820		1.861643
				760	3225		1.868827
761	3834	CHEK1	1.869085
				762	3510	CDK4	1.869212
763	3795	NR4A2	1.870845
				764	3247		1.875796
765	3521	MET	1.887521
				766	3538	NFKB2	1.892227
767	3818		1.900799
				768	3719	BMPR2	1.919267
769	3144	KIF4B	1.924148
				770	3355	GUK1	1.925235
771	2956	PRKCL2	1.929173
				772	3198	ICAM1	1.937953
773	3361	IMPDH2	1.938577
				774	3672	SYK	1.945812
775	3697	CAMK2G	1.946161
				776	3415	HSPCA	1.94686
777	3505	STK6	1.949702
				778	3368	top3A	1.959095
779	3681	SRPK1	1.963919
				780	3613	DVL1	1.984151
781	3720		1.996699
				782	2995	PTK2	2.015836
783	3522	KRAS2	2.026984
				784	3436	BRAF	2.036457
785	3787	FZD4	2.049799
				786	3584	RASAL2	2.089191
787	3098	CENPE	2.090255
				788	3267	CCNH	2.096356
789	2931	MAP4K3	2.11675
				790	2962	MAP4K2	2.12521
791	3790	ERBB3	2.13688
				792	3742	RHOK	2.142917
793	2948	MYO3A	2.173575
				794	3629	AXIN1	2.184253

795	3546	INPP5D	2.197591
				796	3723		2.212338
797	2973	NEK1	2.222767
				798	3512	TGFBR1	2.223853
799	3135		2.223901
				800	3637	STAT4	2.227212
801	3004	MAP3K1	2.235803
				802	3304	CCNE2	2.239326
803	3129	STK6	2.248154
				804	3402	HDAC4	2.253527
805	3627	CTNNA1	2.28197
				806	3537	EIF4EBP1	2.322458
807	3704	ACVR2B	2.322634
				808	3329	CDC42	2.333632
809	3259	MAPK8	2.334959
				810	3689	BLK	2.340679
811	3241	WEE1	2.35419
				812	3137	KIF26A	2.359341
813	3612	TCF1	2.413867
				814	3532		2.468626
815	3764	NOTCH4	2.482525
				816	3417	HDAC3	2.485246
817	3120	PIK3CB	2.528659
				818	3313	CCNG2	2.568855
819	3722	TLK2	2.571781
				820	3136		2.916125
821	3780	MCM3	2.988111
				822	3580	ELK1	3.0307
823	3718	PTK6	3.090027
				824	3777	ABL1	3.099871
825	3605	FZD4	3.155698
				826	3134		3.263194
827	2929	CHUK	3.298485
				828	3781	SRC	3.433423
829	3223		3.587036
				830	3706	C20orf97	4.288466

Table III A is used for the siRNA sequence of the screening of DNA disrupting agent: the cis-platinum screening

The gene title	Serial ID	Adopted sequence is arranged	SEQ?ID?NO
				CHUK	NM_001278	AAAGGCUGCUCACAAGUUCTT	50
CHUK	NM_001278	AGCUGCUCAACAAACCAGATT	51
				CHUK	NM_001278	AUGAGGAACAGGGCAAUAGTT	52
PRKACA	NM_002730	GAAUGGGGUCAACGAUAUCTT	53
				PRKACA	NM_002730	GGACGAGACUUCCUCUUGATT	54
PRKACA	NM_002730	GUGUGGCAAGGAGUUUUCUTT	55
				MAP4K2	NM_004579	GAAUCCUAAGAAGAGGCCGTT	56
MAP4K2	NM_004579	GAGGAGGUCUUUCAUUGGGTT	57
				MAP4K2	NM_004579	GAUAGUCAAGCUAGACCCATT	58
STK17B	NM_004226	AUCCUCCUGUAAUGGAACCTT	59

STK17B	NM_004226	GAAGAGGACAGGAUUGUCGTT	60
				STK17B	NM_004226	GACCAACAGCAGAGAUAUGTT	61
ALK	NM_004304	ACCAGAGACCAAAUGUCACTT	62
				ALK	NM_004304	AUAAGCCCACCAGCUUGUGTT	63
ALK	NM_004304	UCAACACCGCUUUGCCGAUTT	64
				FRK	NM_002031	ACUAUAGACUUCCGCAACCTT	65
FRK	NM_002031	CAGUAGAUUGCUGUGGCCUTT	66
				FRK	NM_002031	CUCCAUACAGCUUCUGAAGTT	67
MAP3K1	AF042838	UCACUUAGCAGCUGAGUCUTT	68
				MAP3K1	AF042838	UUGACAGCACUGGUCAGAGTT	69
MAP3K1	AF042838	UUGGCAAGAACUUCUUGGCTT	70
				KIF2C	NM_006845	ACAAAAACGGAGAUCCGUCTT	71
KIF2C	NM_006845	AUAAGCAGCAAGAAACGGCTT	72
				KIF2C	NM_006845	GAAUUUCGGGCUACUUUGGTT	73
CENPE	NM_001813	GAAAAUGAAGCUUUGCGGGTT	74
				CENPE	NM_001813	GAAGAGAUCCCAGUGCUUCTT	75
CENPE	NM_001813	UCUGAAAGUGACCAGCUCATT	76
				STK6	NM_003600	ACAGUCUUAGGAAUCGUGCTT	3
STK6	NM_003600	GCACAAAAGCUUGUCUCCATT	1
				STK6	NM_003600	UUGCAGAUUUUGGGUGGUCTT	2
KIF4B	AF241316	CCUGCAGCAACUGAUUACCTT	77
				KIF4B	AF241316	GAACUUGAGAAGAUGCGAGTT	78
KIF4B	AF241316	GAAGAGGCCCACUGAAGUUTT	79
				BRCA2	NM_000059	CAAAUGGGCAGGACUCUUATT	80
BRCA2	NM_000059	CUGUUCAGCCCAGUUUGAATT	81
				BRCA2	NM_000059	UCUCCAAGGAAGUUGUACCTT	82
APC	NM_000038	ACCAAGUAUCCGCAAAAGGTT						83
				APC	NM_000038	AGACCUGUAUUAGUACGCCTT	84
APC	NM_000038	CAAGCUUUACCCAGCCUGUTT	85
				ATR	NM_001184	GAAACUGCAGCUAUCUUCCTT	86
ATR	NM_001184	GUUACAAUGAGGCUGAUGCTT	87
				ATR	NM_001184	UCACGACUCGCUGAACUGUTT	88
BRCA1	NM_007296	ACUUAGGUGAAGCAGCAUCTT	89
				BRCA1	NM_007296	GGGCAGUGAAGACUUGAUUTT	90
BRCA1	NM_007296	UGAAGUGGGCUCCAGUAUUTT	91
				DCC	NM_005215	ACAUCGUGGUGCGAGGUUATT	92
DCC	NM_005215	AUGAGCCGCCAAUUGGACATT	93
				DCC	NM_005215	AUGGCAAGUUUGGAAGGACTT	94
WNT1	NM_005430	ACGGCGUUUAUCUUCGCUATT	95
				WNT1	NM_005430	CCCUCUUGCCAUCCUGAUGTT	96
WNT1	NM_005430	CUAUUUAUUGUGCUGGGUCTT	97
				CHEK1	NM_001274	AUCGAUUCUGCUCCUCUAGTT	98
CHEK1	NM_001274	CUGAAGAAGCAGUCGCAGUTT	99
				CHEK1	NM_001274	UGCCUGAAAGAGACUUGUGTT	100
WEE1	NM_003390	AUCGGCUCUGGAGAAUUUGTT	101
				WEE1	NM_003390	CAAGGAUCUCCAGUCCACATT	102
WEE1	NM_003390	UGUACCUGUGUGUCCAUCUTT	103
					NM_018492	AGGACACUUUGGGUACCAGTT	104
	NM_018492	GACCCUAAAGAUCGUCCUUTT	105
					NM_018492	GCUGAGGAGAAUAUGCCUCTT	106
MAPK8	NM_139049	CACCCGUACAUCAAUGUCUTT	107
				MAPK8	NM_139049	GGAAUAGUAUGCGCAGCUUTT	108

MAPK8	NM_139049	GUGAUUCAGAUGGAGCUAGTT	109
				CUL1	NM_003592	GACCGCAAACUACUGAUUCTT	110
CUL1	NM_003592	GCCAGCAUGAUCUCCAAGUTT	111
				CUL1	NM_003592	UAGACAUUGGGUUCGCCGUTT	112
CCNG2	NM_004354	CCUCGAGAAAAAGGGCUGATT	113
				CCNG2	NM_004354	GCUCAGCUGAAAGCUUGCATT	114
CCNG2	NM_004354	UGCCUAGCCGAGUAUUCUUTT	115
				CDC42	NM_044472	ACCUUAUGGAAAAGGGGUGTT	116
CDC42	NM_044472	CCAUCCUGUUUGAAAGCCUTT	117
				CDC42	NM_044472	CCCAAAAGGAAGUGCUGUATT	118
CDC25B	NM_021874	AGGAUGAUGAUGCAGUUCCTT	119
				CDC25B	NM_021874	GACAAGGAGAAUGUGCGCUTT	120
CDC25B	NM_021874	GAGCCCAGUCUGUUGAGUUTT	121
				top2B	NM_001068	ACAUUCCCUGGAGUGUACATT	122
top2B	NM_001068	GAGGAUUUAGCGGCAUUUGTT	123
				top2B	NM_001068	GCUGCUGGACUGCAUAAAGTT	124
IMPDH2	NM_000884	AGAGGGAAGACUUGGUGGUTT	125
				IMPDH2	NM_000884	CACUCAUGCCAGGACAUUGTT	126
IMPDH2	NM_000884	GAAGAAUCGGGACUACCCATT	127
					NM_007027	ACUCACAGAAAAACCGUCGTT	128
	NM_007027	AUGAUGGGCGGACGAGUAUTT	129
					NM_007027	GAGUCAGCACCAUCAAAUGTT	130
HDAC4	NM_006037	AGAGGACGUUUUCUACGGCTT	131
				HDAC4	NM_006037	AUCUGUUUGCAAGGGGAAGTT	132
HDAC4	NM_006037	CAAGAUCAUCCCCAAGCCATT	133
				TERT	NM_003219	CACCAAGAAGUUCAUCUCCTT	134
TERT	NM_003219	GAGUGUCUGGAGCAAGUUGTT	135
				TERT	NM_003219	GUUUGGAAGAACCCCACAUTT	136
BRAF	NM_004333	ACACUUGGUAGACGGGACUTT	137
				BRAF	NM_004333	GUCAAUCAUCCACAGAGACTT	138
BRAF	NM_004333	UUGCAUGUGGAAGUGUUGGTT	139
				ERBB4	NM_005235	GAGUACUCUAUAGUGGCCUTT	140
ERBB4	NM_005235	GCUUCCCAGUCCAAAUGACTT	141
				ERBB4	NM_005235	UGACAGUGGAGCAUGUGUUTT	142
ABL2	NM_007314	AUCAGUGAUGUGGUGCAGATT	143
				ABL2	NM_007314	GACUCGGACACUGAAGAAATT	144
ABL2	NM_007314	UGGCACAGCAGGUACUAAATT	145
				KRAS2	NM_033360	GAAAAGACUCCUGGCUGUGTT	146
KRAS2	NM_033360	GGACUCUGAAGAUGUACCUTT	147
				KRAS2	NM_033360	GGCAUACUAGUACAAGUGGTT	148
	NM_021170	AUCCUGGAGAUGACCGUGATT	149
					NM_021170	GCCGGUCAUGGAGAAGCGGTT	150
	NM_021170	UGGCCCUGAGACUGCAUCGTT	151
				ELK1	NM_005229	GCCAUUCCUUUGUCUGCCATT	152
ELK1	NM_005229	GUGAAAGUAGAAGGGCCCATT	153
				ELK1	NM_005229	UUCAAGCUGGUGGAUGCAGTT	154
RASAL2	NM_004841	AGUACCAGGAUUCUUCAGCTT	155
				RASAL2	NM_004841	CUUAGUUCUGGGCCAUGUATT	156
RASAL2	NM_004841	GACGCCACUGACAGUGAUUTT	157
				ARHGEF2	NM_004723	AGCUACACCACAGAUGCCATT	158
ARHGEF2	NM_004723	GGACUUUGCAGCUGACUCUTT	159
				ARHGEF2	NM_004723	UAAAGGUUGGGGUGGCCAUTT	160

FRAT1	NM_005479	AAGCUAAUGACGAGGAACCTT	161
				FRAT1	NM_005479	CCAUGGUGAAGUGCUUGGATT	162
FRAT1	NM_005479	UAACAGCUGCAAUUCCCUGTT	163
				CTNNA2	NM_004389	CCUGAUGAAUGCUGUUGUCTT	164
CTNNA2	NM_004389	GCACAAUACGGUGACCAAUTT	165
				CTNNA2	NM_004389	UCACAUCUUGGAGGAUGUGTT	166
AXIN1	AF009674	GAAAGUGAGCGACGAGUUUTT	167
				AXIN1	AF009674	GUGCCUUCAACACAGCUUGTT	168
AXIN1	AF009674	UGAAUAUCCAAGAGCAGGGTT	169
				EPHB3	NM_004443	GAAGAUCCUGAGCAGUAUCTT	170
EPHB3	NM_004443	GCUGCAGCAGUACAUUGCUTT	171
				EPHB3	NM_004443	UACCCUGGACAAGCUCAUCTT	172
DDR1	NM_013994	AACAAGAGGACACAAUGGCTT	173
				DDR1	NM_013994	AGAGGUGAAGAUCAUGUCGTT	174
DDR1	NM_013994	UCGCAGACUUUGGCAUGAGTT	175
				CLK2	NM_003993	AUCGUUAGCACCUUAGGAGTT	176
CLK2	NM_003993	CCCCUGCCUUGUACAUAAUTT	177
				CLK2	NM_003993	GUACAAGGAAGCAGCUCGATT	178
C20orf97	NM_021158	AGUCCCAGGUGGGACUCUUTT	179
				C20orf97	NM_021158	CUGGCAUCCUUGAGCUGACTT	180
C20orf97	NM_021158	GACUGUUCUGGAAUGAGGGTT	181
					X95425	ACUGCCAGGAGUAAGAACUTT	182
	X95425	CUAUUACUGCAGAGGGCUUTT	183
					X95425	UGCAUCCUGCAGAGUAUCUTT	184
RPS6KA6	NM_014496	CCUCCUUUCAAACCUGCUUTT	185
				RPS6KA6	NM_014496	GAGGUUCUGUUUACAGAGGTT	186
RPS6KA6	NM_014496	UCAGCCAGUGCAGAUUCAATT	187
					AB002301	AGACAAAGAGGGGACCUUCTT	188
	AB002301	GAAAGUCUAUCCGAAGGCUTT	189
					AB002301	UGCCUCCCUGAAACUUCGATT	190
GPRK6	NM_002082	AAGCAAGAAAUGGCGGCAGTT	191
				GPRK6	NM_002082	GAGCUGAAUGUCUUUGGGCTT	192
GPRK6	NM_002082	UGUAUAUAGCGACCAGAGCTT	193
				GSK3A	NM_019884	CUUCAGUGCUGGUGAACUCTT	194
GSK3A	NM_019884	GCUGGACCACUGCAAUAUUTT	195
				GSK3A	NM_019884	GUGGCUUACACGGACAUCATT	196
RAD51L1	NM_133510	AACAGGACCGUACUGCUUGTT	197
				RAD51L1	NM_133510	GAAGCCUUUGUUCAGGUCUTT	198
RAD51L1	NM_133510	GAGAGGCAUCCUCCUUGAATT	199
				NOTCH4	NM_004557	CCAGCACUGACUACUGUGUTT	200
NOTCH4	NM_004557	GGAACUCGAUGCUUGUCAGTT	201
				NOTCH4	NM_004557	UGCGAGGAAGAUACGGAGUTT	202
MCM3	NM_002388	GCAGAUGAGCAAGGAUGCUTT	203
				MCM3	NM_002388	GUACAUCCAUGUGGCCAAATT	204
MCM3	NM_002388	UGGGUCAUGAAAGCUGCCATT	205
				FZD4	NM_012193	AGAACCUCGGCUACAACGUTT	206
PZD4	NM_012193	UCCGCAUCUCCAUGUGCCATT	207
				FZD4	NM_012193	UCGGCUACAACGUGACCAATT	208

Table III B is used for the siRNA sequence of the screening of DNA disrupting agent: the Zorubicin screening

Gene symbol	Serial ID	Adopted sequence is arranged	?SEQ?ID?NO
				AATK	AB014541	CGCAAGAAGAAGGCCGUGUTT	?209
AATK	AB014541	CGCUGGUGCAAUGUUUUCUTT	?210
				AATK	AB014541	GAAUCCCUACCGAGACUCUTT	?211
ABL1	NM_007313	AAACCUCUACACGUUCUGCTT	?212
				ABL1	NM_007313	CUAAAGGUGAAAAGCUCCGTT	?213
ABL1	NM_007313	UCCUGGCAAGAAAGCUUGATT	?214
				ACVR2	NM_001616	AAGAUGGCCACAAACCUGCTT	?215
ACVR2	NM_001616	AGAUAAACGGCGGCAUUGUTT	?216
				ACVR2	NM_001616	GACAUGCAGGAAGUUGUUGTT	?217
ACVR2B	NM_001106	CGGGAGAUCUUCAGCACACTT	?218
				ACVR2B	NM_001106	GAGAUUGGCCAGCACCCUUTT	?219
ACVR2B	NM_001106	GCCCAGGACAUGAGUGUCUTT	?220
				ADRBK2	NM_005160	CGAGGAUGAGGCAUCUGAUTT	?221
ADRBK2	NM_005160	CUGAAGUCCCUUUUGGAGGTT	?222
				ADRBK2	NM_005160	GAACUUCCCUUUGGUCAUCTT	?223
AKT1	NM_005163	GCUGGAGAACCUCAUGCUGTT	?224
				AKT1	NM_005163	AGACGUUUUUGUGCUGUGGTT	?225
AKT1	NM_005163	CGCACCUUCCAUGUGGAGATT	?226
				AKT2	NM_001626	AGAUGGCCACAUCAAGAUCTT	?227
AKT2	NM_001626	GUCAUCAUUGCCAAGGAUGTT	?228
				AKT2	NM_001626	UGCCAGCUGAUGAAGACCGTT	?229
ALK	NM_004304	ACCAGAGACCAAAUGUCACTT	?230
				ALK	NM_004304	AUAAGCCCACCAGCUUGUGTT	?231
ALK	NM_004304	UCAACACCGCUUUGCCGAUTT	?232
				ALS2CR7	NM_139158	CUGGCUGAUUUUGGUCUUGTT	?233
ALS2CR7	NM_139158	GCCUUCAUGUUGUCUGGAATT	?234
				ALS2CR7	NM_139158	UCCACACCAAAGAGACACUTT	?235
AXIN1	AF009674	GAAAGUGAGCGACGAGUUUTT	?236
				AXIN1	AF009674	GUGCCUUCAACACAGCUUGTT	?237
AXIN1	AF009674	UGAAUAUCCAAGAGCAGGGTT	?238
				BLK	NM_001715	AGUCACGAGCGUUCGAAAATT	?239
BLK	NM_001715	CAACAUGAAGGUGGCCAUUTT	?240
				BLK	NM_001715	GCACUAUAAGAUCCGCUGCTT	?241
BMPR2	NM_001204	CAAAUCUGUGAGCCCAACATT	?242
				BMPR2	NM_001204	CAAGAUGUUCUUGCACAGGTT	?243
BMPR2	NM_001204	GAACGGCUAUGUGCGUUUATT	?244
				BRAF	NM_004333	ACACUUGGUAGACGGGACUTT	?245
BRAF	NM_004333	GUCAAUCAUCCACAGAGACTT	?246
				BRAF	NM_004333	UUGCAUGUGGAAGUGUUGGTT	?247
C20orf97	NM_021158	AGUCCCAGGUGGGACUCUUTT	?248
				C20orf97	NM_021158	CUGGCAUCCUUGAGCUGACTT	?249
C20orf97	NM_021158	GACUGUUCUGGAAUGAGGGTT	?250
				CAMK2G	BC021269	GACAUUGUGGCCAGAGAGUTT	?251
CAMK2G	BC021269	GAUGAGGACCUCAAAGUGCTT	?252
				CAMK2G	BC021269	GGCUGGAGCCUAUGAUUUCTT	?253
CCND3	NM_001760	AAAGCAUGCCCAGACCUUUTT	?254
				CCND3	NM_001760	AAGGAUCUUUGUGGCCAAGTT	?255
CCND3	NM_001760	CUACCUGGAUCGCUACCUGTT	?256
				CCNE2	NM_057749	CCACAGAUGAGGUCCAUACTT	?257
CCNE2	NM_057749	CUGGGGCUUUCUUGACAUGTT	?258
				CCNE2	NM_057749	GUGGUUAAGAAAGCCUCAGTT	?259

CCNG1	NM_004060	AUGGAUUGUUUCUGGGCGUTT	260
				CCNG1	NM_004060	CUAUCAGUCUUCCCACAGCTT	261
CCNG1	NM_004060	CUUGCCACUUGAAAGGAGATT	262
				CCNG2	NM_004354	CCUCGAGAAAAAGGGCUGATT	263
CCNG2	NM_004354	GCUCAGCUGAAAGCUUGCATT	264
				CCNG2	NM_004354	UGCCUAGCCGAGUAUUCUUTT	265
CCNH	NM_001239	GACCCGCUAUCCCAUAUUGTT	266
				CCNH	NM_001239	GCCAGCAAUGCCAAGAUCUTT	267
CCNH	NM_001239	UUGCCCUGACUGCCAUUUUTT	268
				CCNT2	NM_058241	AGCGCCAGUAAAGAAGAACTT	269
CCNT2	NM_058241	AGGGCAGCCAGUUGUCAUUTT	270
				CCNT2	NM_058241	CCACCACUCCAAAAUGAGCTT	271
CDC25B	NM_021874	AGGAUGAUGAUGCAGUUCCTT	272
				CDC25B	NM_021874	GACAAGGAGAAUGUGCGCUTT	273
CDC25B	NM_021874	GAGCCCAGUCUGUUGAGUUTT	274
				CDC42	NM_044472	ACCUUAUGGAAAAGGGGUGTT	275
CDC42	NM_044472	CCAUCCUGUUUGAAAGCCUTT	276
				CDC42	NM_044472	CCCAAAAGGAAGUGCUGUATT	277
CDK3	NM_001258	CGAGAGGAAGCUCUAUCUGTT	278
				CDK3	NM_001258	GAGAGGAUGCAUCUGGGGATT	279
CDK3	NM_001258	GAUCAGACUGGAUUUGGAGTT	280
				CDK4	NM_000075	CAGUCAAGCUGGCUGACUUTT	281
CDK4	NM_000075	GCGAAUCUCUGCCUUUCGATT	282
				CDK4	NM_000075	GGAUCUGAUGCGCCAGUUUTT	283
CDK4	NM_000075	CCCUGGUGUUUGAGCAUGUTT	284
				CDK4	NM_000075	CUGACCGGGAGAUCAAGGUTT	285
CDK4	NM_000075	GAGUGUGAGAGUCCCCAAUTT	286
				CDKN1A	NM_078467	AACUAGGCGGUUGAAUGAGTT	287
CDKN1A	NM_078467	CAUACUGGCCUGGACUGUUTT	288
				CDKN1A	NM_078467	GAUGGUGGCAGUAGAGGCUTT	289
CDKN1C	NM_000076	AAAAACCGGGAUUCCGGCCTT	290
				CDKN1C	NM_000076	GCGCAAGAGAUCAGCGCCUTT	291
CDKN1C	NM_000076	GUGGACAGCGACUCGGUGCTT	292
				CDKN2B	NM_004936	ACACAGAGAAGCGGAUUUCTT	293
CDKN2B	NM_004936	CUCCAAGAGGUGGGUAAUUTT	294
				CDKN2B	NM_004936	UGUCUGCUGAGGAGUUAUGTT	295
CDKN3	NM_005192	CCUGCCUUAAAAAUUACCGTT	296
				CDKN3	NM_005192	GAACUAAAGAGCUGUGGUATT	297
CDKN3	NM_005192	GAGGAUCCGGGGCAAUACATT	298
				CENPE	NM_001813	GAAAAUGAAGCUUUGCGGGTT	299
CENPE	NM_001813	GAAGAGAUCCCAGUGCUUCTT	300
				CENPE	NM_001813	UCUGAAAGUGACCAGCUCATT	301
CHEK1	NM_001274	CCAGUUGAUGUUUGGUCCUTT	302
				CHEK1	NM_001274	UCUCAGACUUUGGCUUGGCTT	303
CHEK1	NM_001274	UUCUAUGGUCACAGGAGAGTT	304
				CHUK	NM_001278	AAAGGCUGCUCACAAGUUCTT	305
CHUK	NM_001278	AGCUGCUCAACAAACCAGATT	306
				CHUK	NM_001278	AUGAGGAACAGGGCAAUAGTT	307
CREBBP	NM_004380	GACAUCCCGAGUCUAUAAGTT	308
				CREBBP	NM_004380	GCACAAGGAGGUCUUCUUCTT	309
CREBBP	NM_004380	UGGAGGAGAAUUAGGCCUUTT	310
				CTNNA1	NM_001903	CGUUCCGAUCCUCUAUACUTT	311

CTNNA1	NM_001903	UGACAUCAUUGUGCUGGCCTT	312
				CTNNA1	NM_001903	UGACCAAAGAUGACCUGUGTT	313
CTNNA2	NM_004389	CCUGAUGAAUGCUGUUGUCTT	314
				CTNNA2	NM_004389	GCACAAUACGGUGACCAAUTT	315
CTNNA2	NM_004389	UCACAUCUUGGAGGAUGUGTT	316
				CTNNAL1	NM_003798	AAGUGUUGUUGCUGGCAGATT	317
CTNNAL1	NM_003798	ACUUGAGAAGCUUUUGGGGTT	318
				CTNNAL1	NM_003798	CUAGAGGUUUUUGCUGCAGTT	319
CUL5	NM_003478	AAGAGUGAGCUGGUCAAUGTT	320
				CUL5	NM_003478	AUUUUGGAGUGCUUGGGCATT	321
CUL5	NM_003478	UGGGUAAACAGGGCAGCAATT	322
				DAPK2	NM_014326	GAAUAUUUUUGGGACGCCGTT	323
DAPK2	NM_014326	UCCAAGAGGCUCUCAGACATT	324
				DAPK2	NM_014326	UCUCAGAAGGUCCUCCUGATT	325
DVL1	NM_004421	GGAGGAGAUCUUUGAUGACTT	326
				DVL1	NM_004421	GUACGCCAGCAGCUUGCUGTT	327
DVL1	NM_004421	UCGGAUCACACGGCACCGATT	328
				DVL3	NM_004423	ACCCCAGUGAGUUCUUUGUTT	329
DVL3	NM_004423	CCUGGACAAUGACACAGAGTT	330
				DVL3	NM_004423	GUUCAUUUAAGCCUCAGGGTT	331
DYRK3	NM_003582	CCAUGUUUGCAUGGCCUUUTT	332
				DYRK3	NM_003582	CUUCUGGAGCAAUCCAAACTT	333
DYRK3	NM_003582	UCUUUGGAUGCCCUCCACATT	334
				ECT2	NM_018098	ACUGGCUAAAGAUGCUGUGTT	335
ECT2	NM_018098	GACCAUGGGAAAAUUGUGGTT	336
				ECT2	NM_018098	GCUUAGUACAGCGGGUUGATT	337
EIF4EBP1	NM_004095	CCACCCCUUCCUUAGGUUGTT	338
				EIF4EBP1	NM_004095	CUCACCUGUGACCAAAACATT	339
EIF4EBP1	NM_004095	UAGCCCAGAAGAUAAGCGGTT	340
				ELK1	NM_005229	GCCAUUCCUUUGUCUGCCATT	341
ELK1	NM_005229	GUGAAAGUAGAAGGGCCCATT	342
				ELK1	NM_005229	UUCAAGCUGGUGGAUGCAGTT	343
EPHB3	NM_004443	GAAGAUCCUGAGCAGUAUCTT	344
				EPHB3	NM_004443	GCUGCAGCAGUACAUUGCUTT	345
EPHB3	NM_004443	UACCCUGGACAAGCUCAUCTT	346
				ERBB3	NM_001982	CUUUCUGAAUGGGGAGCCUTT	347
ERBB3	NM_001982	UACACACACCAGAGUGAUGTT	348
				ERBB3	NM_001982	UGACAGUGGAGCCUGUGUATT	349
ERBB4	NM_005235	GAGUACUCUAUAGUGGCCUTT	350
				ERBB4	NM_005235	GCUUCCCAGUCCAAAUGACTT	351
ERBB4	NM_005235	UGACAGUGGAGCAUGUGUUTT	352
				ERN1	NM_001433	AAGCCUUACGGUCAUGAUGTT	353
ERN1	NM_001433	GAAUAAUGAAGGCCUGACGTT	354
				ERN1	NM_001433	GAUGAUUGCGAUGGAUCCUTT	355
FGFR3	NM_000142	AACAUCAUCAACCUGCUGGTT	356
				FGFR3	NM_000142	CACUUCCAGCAUUUAGCUGTT	357
FGFR3	NM_000142	CACUUCUUACGCAAUGCUUTT	358
				FOXO1A	NM_002015	CUAUGCGUACUGCAUAGCATT	359
FOXO1A	NM_002015	GACAACGACACAUAGCUGGTT	360
				FOXO1A	NM_002015	UACAAGGAACCUCAGAGCCTT	361
FZD4	NM_012193	CCAUCUGCUUGAGCUACUUTT	362
				FZD4	NM_012193	GUUGACUUACCUGACGGACTT	363

FZD4	NM_012193	UUGGCAAAGGCUCCUUGUATT	364
				FZD4	NM_012193	AGAACCUCGGCUACAACGUTT	365
FZD4	NM_012193	UCCGCAUCUCCAUGUGCCATT	366
				FZD4	NM_012193	UCGGCUACAACGUGACCAATT	367
FZD9	NM_003508	GACUUUCCAGACCUGGCAGTT	368
				FZD9	NM_003508	GAUCGGGGUCUUCUCCAUCTT	369
FZD9	NM_003508	GGACUUCGCGCUGGUCUGGTT	370
				GRB2	NM_002086	AUACGUCCAGGCCCUCUUUTT	371
GRB2	NM_002086	CGGGCAGACCGGCAUGUUUTT	372
				GRB2	NM_002086	UGCAGCACUUCAAGGUGCUTT	373
GUCY2D	NM_000180	GAAAUUCCCAGGGGAUCAGTT	374
				GUCY2D	NM_000180	GACAGACCGGCUGCUUACATT	375
GUCY2D	NM_000180	GUCACGGAACUGCAUAGUGTT	376
				GUK1	NM_000858	CGGCAAAGAUUACUACUUUTT	377
GUK1	NM_000858	GGAGCCCGGCCUGUUUGAUTT	378
				GUK1	NM_000858	UCAAGAAAGCUCAAAGGACTT	379
HDAC3	NM_003883	CCCAGAGAUUUUUGAGGGATT	380
				HDAC3	NM_003883	UGCCUUCAACGUAGGCGAUTT	381
HDAC3	NM_003883	UGGUACCUAUUAGGGAUGGTT	382
				HDAC4	NM_006037	AGAGGACGUUUUCUACGGCTT	383
HDAC4	NM_006037	AUCUGUUUGCAAGGGGAAGTT	384
				HDAC4	NM_006037	CAAGAUCAUCCCCAAGCCATT	385
HSPCA	NM_005348	ACACCUGGAGAUAAACCCUTT	386
				HSPCA	NM_005348	CCUAUGGGUCGUGGAACAATT	387
HSPCA	NM_005348	UAACCUUGGUACUAUCGCCTT	388
				ICAM1	NM_000201	CAGCUAAAACCUUCCUCACTT	389
ICAM1	NM_000201	AACACAAAGGCCCACACUUTT	390
				ICAM1	NM_000201	CAGAGUGGAAGACAUAUGCTT	391
1MPDH2	NM_000884	AGAGGGAAGACUUGGUGGUTT	392
				IMPDH2	NM_000884	CACUCAUGCCAGGACAUUGTT	393
IMPDH2	NM_000884	GAAGAAUCGGGACUACCCATT	394
				INPP5D	NM_005541	AGCAUUAAGAAGCCCAGUGTT	395
INPP5D	NM_005541	GAACAAGCACUCAGAGCAGTT	396
				INPP5D	NM_005541	UCCCAUCAACAUGGUGUCCTT	397
IRS2	NM_003749	CACAGCCGUUCGAUGUCCATT	398
				IRS2	NM_003749	GUACAUCGCCAUCGACGUGTT	399
IRS2	NM_003749	GUACCUGAUCGCCCUCUACTT	400
				KIF26A	XM_050278	AUGCGGAAUUUGCCGUGGGTT	401
KIF26A	XM_050278	GCACAAGCACCUGUGUGAGTT	402
				KIF26A	XM_050278	GUCGUACACCAUGAUCGGGTT	403
KIF4B	AF241316	CCUGCAGCAACUGAUUACCTT	404
				KIF4B	AF241316	GAACUUGAGAAGAUGCGAGTT	405
KIF4B	AF241316	GAAGAGGCCCACUGAAGUUTT	406
				KIFB	NM_004521	AAUGCAUCUCGUGAUCGCATT	407
KIF5B	NM_004521	AGACAGUUGGAGGAAUCUGTT	408
				KIF5B	NM_004521	AUCGGCAACUUUAGCGAGUTT	409
KRAS2	NM_033360	GAAAAGACUCCUGGCUGUGTT	410
				KRAS2	NM_033360	GGACUCUGAAGAUGUACCUTT	411
KRAS2	NM_033360	GGCAUACUAGUACAAGUGGTT	412
				MAP2K2	NM_030662	ACCACACCUUCAUCAAGCGTT	413
MAP2K2	NM_030662	AGUCAGCAUCGCGGUUCUCTT	414
				MAP2K2	NM_030662	GAAGGAGAGCCUCACAGCATT	415

MAP3K1	AF042838	UCACUUAGCAGCUGAGUCUTT	416
				MAP3K1	AF042838	UUGACAGCACUGGUCAGAGTT	417
MAP3K1	AF042838	UUGGCAAGAACUUCUUGGCTT	418
				MAP3K4	NM_005922	AGAACGAUCGUCCAGUGGATT	419
MAP3K4	NM_005922	GGUACCUCGAUGCCAUAGUTT	420
				MAP3K4	NM_005922	UUUUGGACUAGUGCGGAUGTT	421
MAP3K5	NM_005923	AGAAUUGGCAGCUGAGUUGTT	422
				MAP3K5	NM_005923	UGCAGCAGCUAUUGCACUUTT	423
MAP3K5	NM_005923	UGUACAGCUUGGAAGGAUGTT	424
				MAP4K2	NM_004579	GAAUCCUAAGAAGAGGCCGTT	425
MAP4K2	NM_004579	GAGGAGGUCUUUCAUUGGGTT	426
				MAP4K2	NM_004579	GAUAGUCAAGCUAGACCCATT	427
MAP4K3	NM_003618	AAUGGGAUGCUGGCAAUGATT	428
				MAP4K3	NM_003618	AUCCUUACACGGGCCAUAATT	429
MAP4K3	NM_003618	CUGGACCUCUGUCAGAACUTT	430
				MAPK8	NM_139049	CACCCGUACAUCAAUGUCUTT	431
MAPK8	NM_139049	GGAAUAGUAUGCGCAGCUUTT	432
				MAPK8	NM_139049	GUGAUUCAGAUGGAGCUAGTT	433
MAPRE1	NM_012325	GAGUAUUAACAGCCUGGACTT	434
				MAPRE1	NM_012325	GCUAAGCUAGAACACGAGUTT	435
MAPRE1	NM_012325	UAGAGGAUGUGUUUCAGCCTT	436
				MARK1	NM_018650	ACAACAGCACUCUUCAGUCTT	437
MARK1	NM_018650	CUGCGAGAGCGAGUUUUACTT	438
				MARK1	NM_018650	UGUGUAUUCUGGAGGUAGCTT	439
MATK	NM_002378	AGCGGAAACACGGGACCAATT	440
				MATK	NM_002378	GUGUGAUGUGACAGCCCAGTT	441
MATK	NM_002378	UGUCACUGAAAGAGGUGUCTT	442
				MCC	NM_002387	AGUUGAGGAGGUUUCUGCATT	443
MCC	NM_002387	GACUUAGAGCUGGGAAUCUTT	444
				MCC	NM_002387	GGAUUAUAUCCAGCAGCUCTT	445
MCM3	NM_002388	GCAGAUGAGCAAGGAUGCUTT	446
				MCM3	NM_002388	GUACAUCCAUGUGGCCAAATT	447
MCM3	NM_002388	UGGGUCAUGAAAGCUGCCATT	448
				MET	NM_000245	AUGCCUCUGGAGUGUAUUCTT	449
MET	NM_000245	AUGCGCCCAUCCUUUUCUGTT	450
				MET	NM_000245	GAUCUGGGCAGUGAAUUAGTT	451
MPHOSPH1	NM_016195	AGAGGAACUCUCUGCAAGCTT	452
				MPHOSPH1	NM_016195	CUGAAGAAGCUACUGCUUGTT	453
MPHOSPH1	NM_016195	GACAUGCGAAUGACACUAGTT	454
				MPHOSPH1	NM_016195	AAGUUUGUGUCCCAGACACTT	455
MPHOSPH1	NM_016195	AAUGGCAGUGAAACACCCUTT	456
				MPHOSPH1	NM_016195	AUGAAGGAGAGUGAUCACCTT	457
MYO3A	NM_017433	AAAGCUACCGAUGUCAGGGTT	458
				MYO3A	NM_017433	AAAUCCCGAGUUAUCCACCTT	459
MYO3A	NM_017433	GGCUAAUGAAAGGUGCUGGTT	460
				NEK1	AB067488	AAGUGACAUUUGGGCUCUGTT	461
NEK1	AB067488	AUGCACGUGCUGCUGUACUTT	462
				NEK1	AB067488	GAAGGACCUUCUGAUUCUGTT	463
NFKB2	NM_002502	AGGAUUCUCAUGGGAAGGGTT	464
				NFKB2	NM_002502	GAAGAACAUGAUGGGGACUTT	465
NFKB2	NM_002502	GAUUGAGCGGCCUGUAACATT	466
				NOTCH1	AF308602	AGGCAAGCCCUGCAAGAAUTT	467

NOTCH1	AF308602	AUAUCGACGAUUGUCCAGGTT	468
				NOTCH1	AF308602	CACUUACACCUGUGUGUGCTT	469
NOTCH3	NM_000435	AAUGGCUUCCGCUGCCUCUTT	470
				NOTCH3	NM_000435	GAACAUGGCCAAGGGUGAGTT	471
NOTCH3	NM_000435	GAGUCUGGGACCUCCUUCUTT	472
				NOTCH4	NM_004557	CCAGCACUGACUACUGUGUTT	473
NOTCH4	NM_004557	GGAACUCGAUGCUUGUCAGTT	474
				NOTCH4	NM_004557	UGCGAGGAAGAUACGGAGUTT	475
NR4A2	NM_006186	AAGGCCGGAGAGGUCGUUUTT	476
				NR4A2	NM_006186	CAUCGACAUUUCUGCCUUCTT	477
NR4A2	NM_006186	GUCACAUGGGCAGAGAUAGTT	478
				NRAS	NM_002524	AAGAGCCACUUUCAAGCUGTT	479
NRAS	NM_002524	AGUAGCAACUGCUGGUGAUTT	480
				NRAS	NM_002524	CCUCUACAGGGAGCAGAUUTT	481
PAK1	NM_002576	CCGCUGUCUCGAUAUGGAUTT	482
				PAK1	NM_002576	GGACCGAUUUUACCGAUCCTT	483
PAK1	NM_002576	UGGAUGGCUCUGUCAAGCUTT	484
				PDGFRB	NM_002609	AAAGAAGUACCAGCAGGUGTT	485
PDGFRB	NM_002609	UCCAUCCACCAGAGUCUAGTT	486
				PDGFRB	NM_002609	UUUGCUGAGCUGCAUCGGATT	487
PDZGEF2	NM_016340	AACCCUCAUCCACAGGUGATT	488
				PDZGEF2	NM_016340	CCGACUGAGUACAUCGAUGTT	489
PDZGEF2	NM_016340	GCCAGAUUCGACUGAUUGUTT	490
				PIK3C2A	NM_002645	AACGAGGAAUCCGACAUUCTT	491
PIK3C2A	NM_002645	UGAUGAGCCCAUCCUUUCATT	492
				PIK3C2A	NM_002645	UGCUUCAACGGAUGUAGCATT	493
PIK3CA	NM_006218	AGGUGCACUGCAGUUCAACTT	494
				PIK3CA	NM_006218	UGGCUUUGAAUCUUUGGCCTT	495
PIK3CA	NM_006218	UUCAGCUAGUACAGGUCCUTT	496
				PIK3CB	NM_006219	AAGUUCAUGUCAGGGCUGGTT	497
PIK3CB	NM_006219	AAUGCGCAAAUUCAGCGAGTT	498
				PIK3CB	NM_006219	CAAAGAUGCCCUUCUGAACTT	499
PKD2	NM_000297	CGGCUAGUACGUGAAGAGUTT	500
				PKD2	NM_000297	CUUAUAGUGGAGCUGGCUATT	501
PKD2	NM_000297	GAUAGCGGACAUAGCUCCATT	502
				PLCG1	NM_002660	ACUACGUGGAAGAGAUGGUTT	503
PLCG1	NM_002660	AGGCAAGAAGUUCCUUCAGTT	504
				PLCG1	NM_002660	GGAGAAUGGUGACCUCAGUTT	505
POLS	NM_006999	ACAGAGACGCCGAAAGUACTT	506
				POLS	NM_006999	CUAGCGACAUAGACCUGGUTT	507
POLS	NM_006999	GCAAAUGAAUUGGCCUGGCTT	508
				PRKACB	NM_002731	AAACCCUUGGAACAGGUUCTT	509
PRKACB	NM_002731	CAAGAUGACAUCUGAGCUCTT	510
				PRKACB	NM_002731	UGUCUGAUCGAUCAUGCAGTT	511
PRKCL1	NM_002741	CACAAGAUCGUCUACAGGGTT	512
				PRKCL1	NM_002741	CACCAGUGAAGUCAGCACUTT	513
PRKCL1	NM_002741	GAUUUCAAGUUCCUGGCGGTT	514
				PRKCL2	NM_006256	AAGUGGCUCCUCAUUGUACTT	515
PRKCL2	NM_006256	AUCAUUCUGGCACCUUCAGTT	516
				PRKCL2	NM_006256	GUAAAAGCGGAAGUAGUCGTT	517
PRKCQ	NM_006257	AAAUGAAGCAAGGCCGCCATT	518
				PRKCQ	NM_006257	ACGGAGUACAGACGUCUCATT	519

PRKCQ	NM_006257	GGAAGCAAAGGACCUUCUGTT	520
				PRKG2	NM_006259	AAGACUGGAUCCUCAGCAGTT	521
PRKG2	NM_006259	CAAGUGCAUCCAGCUGAACTT	522
				PRKG2	NM_006259	GAAAUUCCUGCACAAUGGGTT	523
PRKWNK3	AJ409088	ACCAAGCAGCCAGCUAUACTT	524
				PRKWNK3	AJ409088	CUAAUGACAUCUGGGACCUTT	525
PRKWNK3	AJ409088	CUACGAAGGAAAACGUCAGTT	526
				PRKY	NM_002760	AGACAGUGAAGCUGGUUGUTT	527
PRKY	NM_002760	GAAUUUCUGAGGACGAGCUTT	528
				PRKY	NM_002760	UCAGAUUUGGGCCAGAGUUTT		529
PTEN	NM_000314	UGGAGGGGAAUGCUCAGAATT	530
				PTEN	NM_000314	AAGGCAGCUAAAGGAAGUGTT	531
PTEN	NM_000314	UAAAGAUGGCACUUUCCCGTT	532
				PTK2	NM_005607	CUUGGACGAUGUAUUGGAGTT	533
PTK2	NM_005607	GACUGAAAAUGCUUGGGCATT	534
				PTK2	NM_005607	UCCCACACAUCUUGCUGACTT	535
PTK6	NM_005975	AACACCCUCUGCAAAGUUGTT	536
				PTK6	NM_005975	CCGUGGUUCUUUGGCUGCATT	537
PTK6	NM_005975	UCAGGCUUAUCCGAUGUGCTT	538
				RAB2	NM_002865	AAUUGGCCCUCAGCAUGCUTT	539
RAB2	NM_002865	GAAGGUGAAGCUUUUGCACTT	540
				RAB2	NM_002865	GAGGUUUCAGCCAGUGCAUTT	541
RAD51L1	NM_133510	AACAGGACCGUACUGCUUGTT	542
				RAD51L1	NM_133510	GAAGCCUUUGUUCAGGUCUTT	543
RAD51L1	NM_133510	GAGAGGCAUCCUCCUUGAATT	544
				RAF1	NM_002880	CACUCUCUACCGAAGAUCATT	545
RAF1	NM_002880	GAUCCUAAAGGUUGUCGACTT	546
				RAF1	NM_002880	GGAAGCCAUUUGCAGUGCUTT	547
RASAL2	NM_004841	AGUACCAGGAUUCUUCAGCTT	548
				RASAL2	NM_004841	CUUAGUUCUGGGCCAUGUATT	549
RASAL2	NM_004841	GACCCCACUGACAGUGAUUTT	550
				RASD1	NM_016084	CAAAACCAAGGAGAACGUGTT	551
RASD1	NM_016084	CCUAAGGAGGACCUUUUUGTT	552
				RASD1	NM_016084	GAAACCGUCAUGCCCGCUUTT	553
RHOK	NM_002929	AGUACACAGCAGGUUCAUCTT	554
				RHOK	NM_002929	CGUGAAUGAGGAGAACCCUTT	555
RHOK	NM_002929	GUUUAAGGAGGGGCCUGUGTT	556
				SOS1	NM_005633	AUUGACCACCAGGUUUCUGTT	557
SOS1	NM_005633	CUUACAAAAGGGAGCACACTT	558
				SOS1	NM_005633	UAUCAGACCGGACCUCUAUTT	559
SRC	NM_005417	CAAUUCGUCGGAGGCAUCATT	560
				SRC	NM_005417	GCAGUGCCUGCCUAUGAAATT	561
SRC	NM_005417	GGGGAGUUUGCUGGACUUUTT	562
				SRC	NM_005417	GAACCGGAUGCAGUUGAGCTT	563
SRC	NM_005417	GCCGGAAUACAAGAACGGGTT	564
				SRC	NM_005417	GUGGCUCUUAUCCGCAUGATT	565
SRPK1	NM_003137	CGCUUAUGGAACGUGAUACTT	566
				SRPK1	NM_003137	GCAACAGAAUGGCAGCGAUTT	567
SRPK1	NM_003137	GUUCUAAUCGGAUCUGGCUTT	568
				STAT2	NM_005419	AAAGCCUGCAUCAGAGCUCTT	569
STAT2	NM_005419	AGUUAAUCUCCAGGAACGGTT	570
				STAT2	NM_005419	UUUGCUCAGCCCAAACCUUTT	571

STAT4	NM_003151	ACACAGAUCUGCCUCUAUGTT	572
				STAT4	NM_003151	CCCUACAAUAAAGGCCGGUTT	573
STAT4	NM_003151	UUAGGAAGGUCCUUCAGGGTT	574
				STK10	NM_005990	AGACCAGUACUUCCUCCAGTT	575
STK10	NM_005990	CCAUACUCAGAACUCCUCUTT	576
				STK10	NM_005990	GAAAAAGCAUCAGGGGGAATT	577
STK38L	BC028603	AGACACCUUGACAGAAGAGTT	578
				STK38L	BC028603	CUCUGGGGAUUUCUCAAUGTT	579
STK38L	BC028603	GGAAGUAAAUGCAGGCCAGTT	580
				STK6	NM_003600	ACAGUCUUAGGAAUCGUGCTT	581
STK6	NM_003600	GCACAAAAGCUUGUCUCCATT	582
				STK6	NM_003600	UUGCAGAUUUUGGGUGGUCTT	583
STK6	NM_003600	CACCCAAAAGAGCAAGCAGTT	584
				STK6	NM_003600	CCUCCCUAUUCAGAAAGCUTT	585
STK6	NM_003600	GACUUUGAAAUUGGUCGCCTT	586
				STMN1	NM_005563	AACUGCACAGUGCUGUUGGTT	587
STMN1	NM_005563	UACCCAACGCACAAAUGACTT	588
				STMN1	NM_005563	UGGCUAGUACUGUAUUGGCTT	589
SYK	NM_003177	AGAACUGGGCUCUGGUAAUTT	590
				SYK	NM_003177	AGAAGUUCGACACGCUCUGTT	591
SYK	NM_003177	GGAAAACCUCAUCAGGGAATT	592
				TCF1	NM_000545	AGCCGUGGUGGAGACCCUUTT	593
TCF1	NM_000545	AGUCAAGGAGAAAUGCGGUTT	594
				TCF1	NM_000545	CCUCGUCACGGAGGUGCGUTT	595
TGFBR1	NM_004612	GACAUGAUUCAGCCACAGATT	596
				TGFBR1	NM_004612	UUCCUCGAGAUAGGCCGUUTT	597
TGFBR1	NM_004612	UUUGGGAGGUCAGUUGUUCTT	598
				TGFBR2	NM_003242	CCAGAAAUCCUGCAUGAGCTT	599
TGFBR2	NM_003242	GCAGAACACUUCAGAGCAGTT	600
				TIE	NM_005424	AAAAAGGGAUCUGGGGAUGTT	601
TIE	NM_005424	CGUGACGUUAAUGAACCUGTT	602
				TIE	NM_005424	GAGCAACGGAUCCUACUUCTT	603
TK1	NM_003258	AAGCACAGAGUUGAUGAGATT	604
				TK1	NM_003258	CCUUGCUGGGACUUGGAUCTT	605
TK1	NM_003258	CGCCGGGAAGACCGUAAUUTT	606
				TLE1	NM_005077	AGAUGACAAGAAGCACCACTT	607
TLE1	NM_005077	AGGAAGGUGGAUGAUAAGGTT	608
				TLE1	NM_005077	CACCUGUUUCCAAACCUUGTT	609
TLK2	NM_006852	AGAGCUGGAUCAUCCCAGATT	610
				TLK2	NM_006852	AGGCGUUUAUUCGACGAUGTT	611
TLK2	NM_006852	CACUUGACGGUUGUCCCUUTT	612
				top3A	NM_004618	GAUCCUCCCUGUCUAUGAGTT	613
top3A	NM_004618	GCAAAGAAAUUGGACGAGGTT	614
				top3A	NM_004618	GGCGAAAACAUCGGGUUUGTT	615
TYRO3	NM_006293	GACUAACAAAGGCAGCUGUTT	616
				TYRO3	NM_006293	GCAGCUUGCAUGAAGGAGUTT	617
TYRO3	NM_006293	UGCCCCUUUCCAACUGUCUTT	618
				VHL	NM_000551	AGGAAAUAGGCAGGGUGUGTT	619
VHL	NM_000551	CAGAACCCAAAAGGGUAAGTT	620
				VHL	NM_000551	GAUCUGGAAGACCACCCAATT	621
WASL	NM_003941	AAACAGGAGGUGUUGAAGCTT	622
				WASL	NM_003941	AAGUGGAGCAGAACAGUCGTT	623

WASL	NM_003941	GGACAAUCCACAGAGAUCUTT	624
				WEE1	NM_003390	AUCGGCUCUGGAGAAUUUGTT	625
WEE1	NM_003390	CAAGGAUCUCCAGUCCACATT	626
				WEE1	NM_003390	UGUACCUGUGUGUCCAUCUTT	627
WNT1	NM_005430	ACGGCGUUUAUCUUCGCUATT	628
				WNT1	NM_005430	CCCUCUUGCCAUCCUGAUGTT	629
WNT1	NM_005430	CUAUUUAUUGUGCUGGGUCTT	630
				WNT2	NM_003391	AUUUGCCCGCGCAUUUGUGTT	631
WNT2	NM_003391	AACGGGCGAUUAUCUCUGGTT	632
				WNT2	NM_003391	AGAAGAUGAAUGGUCUGGCTT	633
ZW10	NM_004724	ACAGUUGCAGGAGUUUUCCTT	634
				ZW10	NM_004724	CAAACUGUCAGGCAGCAUUTT	635
ZW10	NM_004724	GCCAGCUUGCAAGAAAUUGTT	636
					XM_170783	ACCGACACUUUGGCUUCCATT	637
	XM_170783	GAUGAGCGCGGGAAUGUUGTT	638
					XM_170783	UGGCCGAGGCCUUCAAGCUTT	639
	XM_064050	CAUCAAUCACUCUCUGCUGTT	640
					XM_064050	CUAACCCAGGAUGUUCAGGTT	641
	XM_064050	GACACUCACCAUGCUGAAATT	642
					XM_066649	AAGGGUGACUUUGUGUCCUTT	643
	XM_066649	ACCAGGAACAAACCUGUUGTT	644
					XM_066649	UUUGAAGGUGGCCCUCCUATT	645
	NM_005200	AUGAAGCCUCACCAGGACUTT	646
					NM_005200	CACUUUUCCCUCAACGAGGTT	647
	NM_005200	UAGUAGCAAAGCAGGAAGGTT	648
					NM_139286	GUUGUAGGAGGCAGUGAUGTT	649
	NM_139286	UCUCAAUUUGGAAGUCUUGTT	650
					NM_139286	UGAUCAAUGAUCGGAUUGGTT	651
	NM_013366	CGAUCUGCAGGCCAACAUCTT	652
					NM_013366	GAAGUAUGAGCAGCUCAAGTT	653
	NM_013366	GGACCUCUUCAUCAAUGAGTT	654
					NM_014885	ACCAGGAUUUGGAGUGGAUTT	655
	NM_014885	CAAGGCAUCCGUUAUAUCUTT	656
					NM_014885	GUGGCUGGAUUCAUGUUCCTT	657
	NM_016263	CCAGAUCCUUGUCUGGAAGTT	658
					NM_016263	CGACAACAAGCUGCUGGUCTT	659
	NM_016263	GAAGCUGUCCAUGUUGGAGTT	660
					NM_013367	AGCCAGCAGAUGUAAUUGGTT	661
	NM_013367	CAUUUCAAUGAGGCUCCAGTT	662
					NM?013367	GUCAUUUACAGAGUGGCUCTT	663
	NM_018492	AGGACACUUUGGGUACCAGTT	664
					NM_018492	GACCCUAAAGAUCGUCCUUTT	665
	NM_018492	GCUGAGGAGAAUAUGCCUCTT	666
					NM_006087	CGUGUACUACAACGAGGCCTT	667
	NM_006087	UCCCCUCUGACUCCAACUUTT	668
					NM_006087	CGAGGCACUCUACGACAUCTT	669
	NM_016231	GCAAUGAGGACAGCUUGUGTT	670
					NM_016231	UCUCCUUGUGAACAGCAACTT	671
	NM_016231	UGUAGCUUUCCACUGGAGUTT	672
					XM_095827	AAGGUCUUUACGCCAGUACTT	673
	XM_095827	GGAAUGUAUCCGAGCACUGTT	674
					XM_095827	UAAGCCUGGUGGUGAUCUUTT	675

	NM_145754	CUCAAGGGAAAUAUCCGUGTT	676
					NM_145754	GUGUGUUGUGCCUGCUGAATT	677
	NM_145754	UCAGGCAUGGCAUUAAAACTT	678
					XM_168069	CAAAGUUAUUAGCCCCAAGTT	679
	XM_168069	CAGAGGCCAAGUAUAUCAATT	680
					XM_168069	CCUGCAGAUUUGCACAGCGTT	681
	NM_021170	AUCCUGGAGAUGACCGUGATT	682
					NM_021170	GCCGGUCAUGGAGAAGCGGTT	683
	NM_021170	UGGCCCUGAGACUGCAUCGTT	684
					NM_019089	CCCCUCCAUGCUCAGAACUTT	685
	NM_019089	CCUAUCUGGGAAGCCUGUGTT	686
					NM_019089	UGCCCCAGUGACAAUAACATT	687
	NM_016653	ACCAGGGCCAAAAUUAUGGTT						688
					NM_016653	AUAGUGAACCUGGAACUGGTT	689
	NM_016653	GCAGUUGCCCCAGAAGUUUTT	690
					NM_016281	AGAACACACUGCUUGGUUGTT	691
	NM_016281	GACAGUGAACAUGGAACCATT	692
					NM_016281	GAGAACUUGCAGCACACACTT	693
	NM_012119	ACCUGCCAACCUGCUCAUCTT	694
					NM_012119	GAUCUCCUUUAAGGAGCAGTT	695
	NM_012119	GCAGCUGUGUAUUUAAGGATT	696
					AB002301	AGACAAAGAGGGGACCUUCTT	697
	AB002301	GAAAGUCUAUCCGAAGGCUTT	698
					AB002301	UGCCUCCCUGAAACUUCGATT	699
	NM_018401	AGGUAUGCAUCGUGCAGAATT	700
					NM_018401	GCAAUCAAACCGUCAUGACTT	701
	NM_018401	UAUCCUGCUGGAUGAACACTT	702
					NM_006622	GCAAGGUAUACAAUGCCGUTT	703
	NM_006622	UAACUCAGCAACCCAGCAATT	704
					NM_006622	UGCCUUGAAGACAGUACCATT	705
	AI278633	CCUCAGCCGUAUAAUACGUTT	706
					AI278633	CUGCUCUGUUCAAUCCCAGTT	707
	AI278633	CUGGGAUUGGCCACCUCUUTT	708
					NM_152524	AGAAGGAGAGUGUCAGGUUTT	709
	NM_152524	UAUGUACCCCGUUCAGCAATT	710
					NM_152524	UUUUGCCUUGGAGUGCUCCTT	711
	NM_019013	AAAACCCCCGGGAGUCGUCTT	712
					NM_019013	AGUGGCACCAAGUGGCUGGTT	713
	NM_019013	GAAACCUGCUUUGUCAUUUTT	714
					AI338451	CUGAUGCACUUUGCUGCAGTT	715
	AI338451	CUGCAGGUUCAAAUCCCAGTT	716
					AI338451	GGGGAAAAAGCUUUGCGUUTT	717
	NM_018410	AAAGACCCAGGCUAUCAGATT	718
					NM_018410	CAGACCCCAAAUCCAUAAGTT	719
	NM_018410	GUCAGUGUCACCCAGCAAATT	720
					NM_018123	UAUCGAGCCACCAUUUGUGTT	721
	NM_018123	UGAUGCAUAUAGCCGCAACTT	722
					NM_018123	UGCACAGGGCCAAAGUUGATT	723

Table III C is used for the siRNA sequence of the screening of DNA disrupting agent: the camptothecine screening

Gene symbol	Serial ID	Adopted sequence is arranged	SEQ?ID?NO
				AATK	AB014541	CGCAAGAAGAAGGCCGUGUTT	724
AATK	AB014541	CGCUGGUGCAAUGUUUUCUTT	725
				AATK	AB014541	GAAUCCCUACCGAGACUCUTT	726
ABL1	NM_007313	AAACCUCUACACGUUCUGCTT	727
				ABL1	NM_007313	CUAAAGGUGAAAAGCUCCGTT	728
ABL1	NM_007313	UCCUGGCAAGAAAGCUUGATT	729
				ABL2	NM_007314	AUCAGUGAUGUGGUGCAGATT	730
ABL2	NM_007314	GACUCGGACACUGAAGAAATT	731
				ABL2	NM_007314	UGGCACAGCAGGUACUAAATT	732
ACVR2	NM_001616	AAGAUGGCCACAAACCUGCTT	733
				ACVR2	NM_001616	AGAUAAACGGCGGCAUUGUTT	734
ACVR2	NM_001616	GACAUGCAGGAAGUUGUUGTT	735
				ACVR2B	NM_001106	CGGGAGAUCUUCAGCACACTT	736
ACVR2B	NM_001106	GAGAUUGGCCAGCACCCUUTT	737
				ACVR2B	NM_001106	GCCCAGGACAUGAGUGUCUTT	738
AKT2	NM_001626	AGAUGGCCACAUCAAGAUCTT	739
				AKT2	NM_001626	GUCAUCAUUGCCAAGGAUGTT	740
AKT2	NM_001626	UGCCAGCUGAUGAAGACCGTT	741
				ANAPC5	NM_016237	ACAGUGCUGAACUUGGCUUTT	742
ANAPC5	NM_016237	CCAAAUGUCAGAGGCACAUTT	743
				ANAPC5	NM_016237	UCAAACUGAUGGCUGAAGGTT	744
AXIN1	AF009674	GAAAGUGAGCGACGAGUUUTT	745
				AXIN1	AF009674	GUGCCUUCAACACAGCUUGTT	746
AXIN1	AF009674	UGAAUAUCCAAGAGCAGGGTT	747
				BCL2	NM_000633	AGGACAUUUGUUGGAGGGGTT	748
BCL2	NM_000633	GCUACCAAUUGUGCCGAGATT	749
				BCL2	NM_000633	UGAAGAACGUGGACGCUUUTT	750
BLK	NM_001715	AGUCACGAGCGUUCGAAAATT	751
				BLK	NM_001715	CAACAUGAAGGUGGCCAUUTT	752
BLK	NM_001715	GCACUAUAAGAUCCGCUGCTT	753
				BMPR1B	NM_001203	ACAGAUUGGAAAAGGUCGCTT	754
BMPR1B	NM_001203	GAAGUUACGCCCCUCAUUCTT	755
				BMPR1B	NM_001203	UAUUUGCAGCACAGACGGATT	756
BMPR2	NM_001204	CAAAUCUGUGAGCCCAACATT	757
				BMPR2	NM_001204	CAAGAUGUUCUUGCACAGGTT	758
BMPR2	NM_001204	GAACGGCUAUGUGCGUUUATT	759
				BRCA1	NM_007296	ACUUAGGUGAAGCAGCAUCTT	760
BRCA1	NM_007296	GGGCAGUGAAGACUUGAUUTT	761
				BRCA1	NM_007296	UGAAGUGGGCUCCAGUAUUTT	762
BRCA2	NM_000059	CAAAUGGGCAGGACUCUUATT	763
				BRCA2	NM_000059	CUGUUCAGCCCAGUUUGAATT	764
BRCA2	NM_000059	UCUCCAAGGAAGUUGUACCTT	765
				C20orf97	NM_021158	AGUCCCAGGUGGGACUCUUTT	766
C20orf97	NM_021158	CUGGCAUCCUUGAGCUGACTT	767
				C20orf97	NM_021158	GACUGUUCUGGAAUGAGGGTT	768
CAMK2D	NM_001221	ACCAGAUGGAGUAAAGGAGTT	769
				CAMK2D	NM_001221	GCACCCUAAUAUUGUGCGATT	770
CAMK2D	NM_001221	UUGGCAGACUUUGGCUUAGTT	771
				CCND1	NM_053056	CAUGUAACCGGCAUGUUUCTT	772
CCND1	NM_053056	CCCACAGCUACUUGGUUUGTT	773
				CCND1	NM_053056	UGACCCCGCACGAUUUCAUTT	774

CCNE2	NM_057749	CCACAGAUGAGGUCCAUACTT	775
				CCNE2	NM_057749	CUGGGGCUUUCUUGACAUGTT	776
CCNE2	NM_057749	GUGGUUAAGAAAGCCUCAGTT	777
				CCNT2	NM_058241	AGCGCCAGUAAAGAAGAACTT	778
CCNT2	NM_058241	AGGGCAGCCAGUUGUCAUUTT	779
				CCNT2	NM_058241	CCACCACUCCAAAAUGAGCTT	780
CDC14B	NM_033331	GGCCAUCCCCUCCAUUAAUTT	781
				CDC14B	NM_033331	GUAAUUGAAAGGCAGUGCCTT	782
CDC14B	NM_033331	UUGCUAUCACUGUGGCUCUTT	783
				CDC16	NM_003903	AGUGGCUUCAAAGAUCCCUTT	784
CDC16	NM_003903	GCAUGUCGUUACCUUGCAGTT	785
				CDC16	NM_003903	UAAGCCUAGUGAAACGGUCTT	786
CDC23	NM_004661	AGCAACUGCUGCUUAUUGCTT	787
				CDC23	NM_004661	AGCAAGCAAGGAGAUAGGATT	788
CDC23	NM_004661	CCUUCUUUAUGUCAGGAGCTT	789
				CDC25B	NM_021874	AGGAUGAUGAUGCAGUUCCTT	790
CDC25B	NM_021874	GACAAGGAGAAUGUGCGCUTT	791
				CDC25B	NM_021874	GAGCCCAGUCUGUUGAGUUTT	792
CDC34	NM_004359	ACGUGGACGCCUCCGUGAUTT	793
				CDC34	NM_004359	CACCUACUACGAGGGCGGCTT	794
CDC34	NM_004359	CAUCUACGAGACGGGGGACTT	795
				CDC37	NM_007065	CGCAUGGAGCAGUUCCAGATT	796
CDC37	NM_007065	GACGGCUUCAGCAAGAGCATT	797
				CDC37	NM_007065	GAUUAAGACAGCCGAUCGCTT	798
CDC42	NM_044472	ACCUUAUGGAAAAGGGGUGTT	799
				CDC42	NM_044472	CCAUCCUGUUUGAAAGCCUTT	800
CDC42	NM_044472	CCCAAAAGGAAGUGCUGUATT	801
				CDC45L	NM_003504	CACCUGCUCAAGUCCUUUGTT	802
CDC45L	NM_003504	GAUCCUUCAGGCCUUGUUCTT	803
				CDC45L	NM_003504	UGACAGUGAUGGGUCAGAGTT	804
CDK2	NM_001798	AUGAUAGCGGGGGCUAAGUTT	805
				CDK2	NM_001798	GAGCUAUCUGUUCCAGCUGTT	806
CDK2	NM_001798	UCUAUUGCUUCACCAUGGCTT	807
				CDK2AP1	NM_004642	AGCAAAUACGCGGAGCUGCTT	808
CDK2AP1	NM_004642	CUGCCCAGGUUUUUUUUGUTT	809
				CDK2AP1	NM_004642	GUUACAGUUCAUCUCCCCUTT	810
CDK4	NM_000075	CCCUGGUGUUUGAGCAUGUTT	811
				CDK4	NM_000075	CUGACCGGGAGAUCAAGGUTT	812
CDK4	NM_000075	GAGUGUGAGAGUCCCCAAUTT	813
				CDK5R2	NM_003936	AGGCGAGAGCCGACUCAAGTT	814
CDK5R2	NM_003936	CCUGGACCGCUAGGGAUACTT	815
				CDK5R2	NM_003936	CGCAACCGCGAGAACCUUCTT	816
CDK7	NM_001799	AACUGGCAGAUUUUGGCCUTT	817
				CDK7	NM_001799	CUGUCCAGUGGAAACCUUATT	818
CDK7	NM_001799	UAGAACCGCCUUAAGAGAGTT	819
				CDKL5	NM_003159	ACAGUACCCAAUUCCGACATT	820
CDKL5	NM_003159	GGAGAAUACUUCUGCUGUGTT	821
				CDKL5	NM_003159	UCAGCCACAAUGAUGUCCUTT	822
CDKN1A	NM_078467	AACUAGGCGGUUGAAUGAGTT	823
				CDKN1A	NM_078467	CAUACUGGCCUGGACUGUUTT	824
CDKN1A	NM_078467	GAUGGUGGCAGUAGAGGCUTT	825
				CHEK1	NM_001274	AUCGAUUCUGCUCCUCUAGTT	826

CHEK1	NM_001274	CUGAAGAAGCAGUCGCAGUTT	827
				CHEK1	NM_001274	UGCCUGAAAGAGACUUGUGTT	828
CHEK1	NM_001274	CCAGUUGAUGUUUGGUCCUTT	829
				CHEK1	NM_001274	UCUCAGACUUUGGCUUGGCTT	830
CHEK1	NM_001274	UUCUAUGGUCACAGGAGAGTT	831
				CHFR	NM_018223	AGACUGCGUCCUUUUCGUCTT	832
CHFR	NM_018223	GAUACCAGCACCAGUGGAATT	833
				CHFR	NM_018223	GCAUACCUCAUCCAGCAUCTT	834
CKAP2	NM_018204	CCAAUCACAAGUCCUAUUGTT	835
				CKAP2	NM_018204	CUUGUGCGACCUCCUAUUATT	836
CKAP2	NM_018204	GAGAGAAAAGCUCGUCUGATT	837
				CREBBP	NM_004380	AUUUUUGCGGCGCCAGAAUTT	838
CREBBP	NM_004380	GAAAAACGGAGGUCGCGUUTT	839
				CREBBP	NM_004380	GAAAACAAAUGCCCCGUGCTT	840
CSF1R	NM_005211	AGUGCAGAAAGUCAUCCCATT	841
				CSF1R	NM_005211	CAACCUGCAGUUUGGUAAGTT	842
CSF1R	NM_005211	UGAGCCAAGUGGCAGCUAATT	843
				CTNNA1	NM_001903	CGUUCCGAUCCUCUAUACUTT	844
CTNNA1	NM_001903	UGACAUCAUUGUGCUGGCCTT	845
				CTNNA1	NM_001903	UGACCAAAGAUGACCUGUGTT	846
CTNNAL1	NM_003798	AAGUGUUGUUGCUGGCAGATT	847
				CTNNAL1	NM_003798	ACUUGAGAAGCUUUUGGGGTT	848
CTNNAL1	NM_003798	CUAGAGGUUUUUGCUGCAGTT	849
				CTNNBIP1	NM_020248	AAAUUUGCGCCUCGGUAUCTT	850
CTNNBIP1	NM_020248	ACCUAAGUCCUUCCACCUGTT	851
				CTNNBIP1	NM_020248	CACCCUGGAUGCUGUUGAATT	852
CUL1	NM_003592	GACCGCAAACUACUGAUUCTT	853
				CUL1	NM_003592	GCCAGCAUGAUCUCCAAGUTT	854
CUL1	NM_003592	UAGACAUUGGGUUCGCCGUTT	855
				DAPK2	NM_014326	GAAUAUUUUUGGGACGCCGTT	856
DAPK2	NM_014326	UCCAAGAGGCUCUCAGACATT	857
				DAPK2	NM_014326	UCUCAGAAGGUCCUCCUGATT	858
DCC	NM_005215	ACAUCGUGGUGCGAGGUUATT	859
				DCC	NM_005215	AUGAGCCGCCAAUUGGACATT	860
DCC	NM_005215	AUGGCAAGUUUGGAAGGACTT	861
				DDR1	NM_013994	AACAAGAGGACACAAUGGCTT	862
DDR1	NM_013994	AGAGGUGAAGAUCAUGUCGTT	863
				DDR1	NM_013994	UCGCAGACUUUGGCAUGAGTT	864
DMPK	NM_004409	CAAGUGGGACAUGCUGAAGTT	865
				DMPK	NM_004409	UAAAAGGCCCUCCAUCUGCTT	866
DMPK	NM_004409	UUGGCCCUGUUCAGCAAUGTT	867
				DTX1	NM_004416	AACCCACCUGAUGAGGACUTT	868
DTX1	NM_004416	GACCGAGUUUGGAUCCAACTT	869
				DTX1	NM_004416	GAUGGAGUUCCACCUCAUCTT	870
DYRK3	NM_003582	CCAUGUUUGCAUGGCCUUUTT	871
				DYRK3	NM_003582	CUUCUGGAGCAAUCCAAACTT	872
DYRK3	NM_003582	UCUUUGGAUGCCCUCCACATT	873
				ECT2	NM_018098	ACUGGCUAAAGAUGCUGUGTT	874
ECT2	NM_018098	GACCAUGGGAAAAUUGUGGTT	875
				ECT2	NM_018098	GCUUAGUACAGCGGGUUGATT	876
EGR2	NM_000399	CACUACCACCCUUUCCUGUTT	877
				EGR2	NM_000399	GUGCAAUGUGAUGGGAGGATT	878

EGR2	NM_000399	UGUUACCGGAGCUGAUUUGTT	879
				ELK1	NM_005229	GCCAUUCCUUUGUCUGCCATT	880
ELK1	NM_005229	GUGAAAGUAGAAGGGCCCATT	881
				ELK1	NM_005229	UUCAAGCUGGUGGAUGCAGTT	882
ELK1	NM_005229	AGGACCCUUUCAAUGUCCCTT	883
				ELK1	NM_005229	CUCUCAUUAUCUCCUCCACTT	884
ELK1	NM_005229	GCUCUCCUUCCAGUUUCCATT	885
				EPHA4	NM_004438	CUGGCUACGAACUGAUUGGTT	886
EPHA4	NM_004438	GAUUCCUAUCCGGUGGACUTT	887
				EPHA4	NM_004438	GCUAUCGUAUAGUUCGGACTT	888
EPHB3	NM_004443	GAAGAUCCUGAGCAGUAUCTT	889
				EPHB3	NM_004443	GCUGCAGCAGUACAUUGCUTT	890
EPHB3	NM_004443	UACCCUGGACAAGCUCAUCTT	891
				ETS1	NM_005238	UUCAGCCUGAAAGGUGUAGTT	892
ETS1	NM_005238	ACGCUACGUGUACCGCUUUTT	893
				ETS1	NM_005238	UGACUACCCCUCGGUCAUUTT	894
FLT1	NM_002019	ACAUCGAAAACAGCAGGUGTT	895
				FLT1	NM_002019	AGGAGGAGUGCAUCUUUGGTT	896
FLT1	NM_002019	UGGAUGAGGACUUUUGCAGTT	897
				FOXO1A	NM_002015	CUAUGCGUACUGCAUAGCATT	898
FOXO1A	NM_002015	GACAACGACACAUAGCUGGTT	899
				FOXO1A	NM_002015	UACAAGGAACCUCAGAGCCTT	900
FRAT1	NM_005479	AAGCUAAUGACGAGGAACCTT	901
				FRAT1	NM_005479	CCAUGGUGAAGUGCUUGGATT	902
FRAT1	NM_005479	UAACAGCUGCAAUUCCCUGTT	903
				FRK	NM_002031	ACUAUAGACUUCCGCAACCTT	904
FRK	NM_002031	CAGUAGAUUGCUGUGGCCUTT	905
				FRK	NM_002031	CUCCAUACAGCUUCUGAAGTT	906
FZD9	NM_003508	GACUUUCCAGACCUGGCAGTT	907
				FZD9	NM_003508	GAUCGGGGUCUUCUCCAUCTT	908
FZD9	NM_003508	GGACUUCGCGCUGGUCUGGTT	909
				GPRK6	NM_002082	AAGCAAGAAAUGGCGGCAGTT	910
GPRK6	NM_002082	GAGCUGAAUGUCUUUGGGCTT	911
				GPRK6	NM_002082	UGUAUAUAGCGACCAGAGCTT	912
GUK1	NM_000858	CGGCAAAGAUUACUACUUUTT	913
				GUK1	NM_000858	GGAGCCCGGCCUGUUUGAUTT	914
GUK1	NM_000858	UCAAGAAAGCUCAAAGGACTT	915
				HDAC3	NM_003883	CCCAGAGAUUUUUGAGGGATT	916
HDAC3	NM_003883	UGCCUUCAACGUAGGCGAUTT	917
				HDAC3	NM_003883	UGGUACCUAUUAGGGAUGGTT	918
HDAC4	NM_006037	AGAGGACGUUUUCUACGGCTT	919
				HDAC4	NM_006037	AUCUGUUUGCAAGGGGAAGTT	920
HDAC4	NM_006037	CAAGAUCAUCCCCAAGCCATT	921
				HDAC5	NM_005474	AAACUGUUCUCAGAUGCCCTT	922
HDAC5	NM_005474	CCCAACUUGAAAGUGCGUUTT	923
				HDAC5	NM_005474	UGAGAUGCACUCCUCCAGUTT	924
HDAC9	NM_058176	AAGCUUCUUGUAGCUGGUGTT	925
				HDAC9	NM_058176	AUAUUGCCUGGACAGGUGGTT	926
HDAC9	NM_058176	CAGCAACAAGAACUCCUAGTT	927
				HSPCB	NM_007355	AGCAUUCAUGGAGGCUCUUTT	928
HSPCB	NM_007355	AUUGACAUCAUCCCCAACCTT	929
				HSPCB	NM_007355	CUCAGCUUUUGUGGAGCGATT	930

IRS1	NM_005544	AGGGCAGUGGAGACUAUAUTT	931
				IRS1	NM_005544	CCAGAGUGCCAAAGUGAUCTT	932
IRS1	NM_005544	GGAUAUAUUUGGCUGGGUGTT	933
				KIF17	XM_027915	GAUAACGGCUUCUGGAAGATT	934
KIF17	XM_027915	GCAAAAGCAACUUUGGCAGTT	935
				KIF17	XM_027915	GCUCAAUAUCAGCUGGGAATT	936
KIF25	NM_005355	GAGCUAUACCAUGCUGGGATT	937
				KIF25	NM_005355	GGAUGGACGGACAGAGGUUTT	938
KIF25	NM_005355	GUUACUGGUGAUUCUCUGCTT	939
				KIF26A	XM_050278	AUGCGGAAUUUGCCGUGGGTT	940
KIF26A	XM_050278	GCACAAGCACCUGUGUGAGTT	941
				KIF26A	XM_050278	GUCGUACACCAUGAUCGGGTT	942
KIF2C	NM_006845	ACAAAAACGGAGAUCCGUCTT	943
				KIF2C	NM_006845	AUAAGCAGCAAGAAACGGCTT	944
KIF2C	NM_006845	GAAUUUCGGGCUACUUUGGTT	945
				KIF3B	NM_004798	AAACGGUCCAUUGGUAGGATT	946
KIF3B	NM_004798	AAGUGGAAGGAAGUCGGGATT	947
				KIF3B	NM_004798	UGCCAAGCAGUUUGAACUGTT	948
KIF4B	AF241316	CCUGCAGCAACUGAUUACCTT	949
				KIF4B	AF241316	GAACUUGAGAAGAUGCGAGTT	950
KIF4B	AF241316	GAAGAGGCCCACUGAAGUUTT	951
				KRAS2	NM_033360	GAAAAGACUCCUGGCUGUGTT	952
KRAS2	NM_033360	GGACUCUGAAGAUGUACCUTT	953
				KRAS2	NM_033360	GGCAUACUAGUACAAGUGGTT	954
LATS2	NM_014572	AACAGCCAUCCAAGUCUUCTT	955
				LATS2	NM_014572	AACCUACCAGCAGAAGGUUTT	956
LATS2	NM_014572	UAGGCUUUUCAGGACCUUCTT	957
				MAP2K7	NM_005043	AGUCCUACAGGAAGAGCCCTT	958
MAP2K7	NM_005043	GCUACUUGAACACAGCUUCTT	959
				MAP2K7	NM_005043	UCAACGACCUGGAGAACUUTT	960
MAP3K1	AF042838	UCACUUAGCAGCUGAGUCUTT	961
				MAP3K1	AF042838	UUGACAGCACUGGUCAGAGTT	962
MAP3K1	AF042838	UUGGCAAGAACUUCUUGGCTT	963
				MAP3K4	NM_005922	AGAACGAUCGUCCAGUGGATT	964
MAP3K4	NM_005922	GGUACCUCGAUGCCAUAGUTT	965
				MAP3K4	NM_005922	UUUUGGACUAGUGCGGAUGTT	966
MAP4K5	NM_006575	AAGGCUGCCACAAAUGUUGTT	967
				MAP4K5	NM_006575	GAAACAGAAGCACGAGAUGTT	968
MAP4K5	NM_006575	UCUCUACAUCUUGGCUGGATT	969
				MAPK13	NM_002754	CUCACAGUGGAUGAAUGGATT	970
MAPK13	NM_002754	GAUCAUGGGGAUGGAGUUCTT	971
				MAPK13	NM_002754	UACAGCCUUUCAAGCAGAGTT	972
MAPK8	NM_139049	CACCCGUACAUCAAUGUCUTT	973
				MAPK8	NM_139049	GGAAUAGUAUGCGCAGCUUTT	974
MAPK8	NM_139049	GUGAUUCAGAUGGAGCUAGTT	975
				MAPRE1	NM_012325	GAGUAUUAACAGCCUGGACTT	976
MAPRE1	NM_012325	GCUAAGCUAGAACACGAGUTT	977
				MAPRE1	NM_012325	UAGAGGAUGUGUUUCAGCCTT		978
MAPRE3	NM_012326	CAGCUUUGUUCAGGGGCAGTT	979
				MAPRE3	NM_012326	CUUCGUGACAUCGAGCUCATT	980
MAPRE3	NM_012326	GGAUUACAACCCUCUGCUGTT	981
				MARK1	NM_018650	ACAACAGCACUCUUCAGUCTT	982

MARK1	NM_018650	CUGCGAGAGCGAGUUUUACTT	983
				MARK1	NM_018650	UGUGUAUUCUGGAGGUAGCTT	984
MCC	NM_002387	AGUUGAGGAGGUUUCUGCATT	985
				MCC	NM_002387	GACUUAGAGCUGGGAAUCUTT	986
MCC	NM_002387	GGAUUAUAUCCAGCAGCUCTT	987
				MCM3	NM_002388	AGGAUUUUGUGGCCUCCAUTT	988
MCM3	NM_002388	GUCUCAGCUUCUGCGGUAUTT	989
				MCM3	NM_002388	UCCAGGUUGAAGGCAUUCATT	990
MCM3	NM_002388	GCAGAUGAGCAAGGAUGCUTT	991
				MCM3	NM_002388	GUACAUCCAUGUGGCCAAATT	992
MCM3	NM_002388	UGGGUCAUGAAAGCUGCCATT	993
				MLH1	NM_000249	AACUGAAAGCCCCUCCUAATT	994
MLH1	NM_000249	GAUGGAAAUAUCCUGCAGCTT	995
				MLH1	NM_000249	UGCUGUUAGUCGAGAACUGTT	996
MYB	NM_005375	ACAAGAGGUGGAAUCUCCATT	997
				MYB	NM_005375	GGUUAUCUGCAGGAGUCUUTT	998
MYB	NM_005375	UCGAACAGAUGUGCAGUGCTT	999
				MYO3A	NM_017433	AAAGCUACCGAUGUCAGGGTT	1000
MYO3A	NM_017433	AAAUCCCGAGUUAUCCACCTT	1001
				MYO3A	NM_017433	GGCUAAUGAAAGGUGCUGGTT	1002
NEK1	AB067488	AAGUGACAUUUGGGCUCUGTT	1003
				NEK1	AB067488	AUGCACGUGCUGCUGUACUTT	1004
NEK1	AB067488	GAAGGACCUUCUGAUUCUGTT	1005
				NF1	NM_000267	AUCCUUCAACAAGGCACAGTT	1006
NF1	NM_000267	GUAACUUCAGCAGAGCGAATT	1007
				NF1	NM_000267	UACAUGACUCCAUGGCUGUTT	1008
NFKB2	NM_002502	AGGAUUCUCAUGGGAAGGGTT	1009
				NFKB2	NM_002502	GAAGAACAUGAUGGGGACUTT	1010
NFKB2	NM_002502	GAUUGAGCGGCCUGUAACATT	1011
				NTRK1	NM_002529	CAACGGCAACUACACGCUGTT	1012
NTRK1	NM_002529	CGCCACAGCAUCAAGGAUGTT	1013
				NTRK1	NM_002529	GAGUGGUCUCCGUUUCGUGTT	1014
OSR1	NM_005109	GAUACACAAAGAUGGGCUGTT	1015
				OSR1	NM_005109	AAACAGCUCAGGCUUUGUCTT	1016
OSR1	NM_005109	GAAUAGUGGCUUACCGCUUTT	1017
				PAK1	NM_002576	CCGCUGUCUCGAUAUGGAUTT	1018
PAK1	NM_002576	GGACCGAUUUUACCGAUCCTT	1019
				PAK1	NM_002576	UGGAUGGCUCUGUCAAGCUTT	1020
PCNA	NM_002592	AAUUGCGGAUAUGGGACACTT	1021
				PCNA	NM_002592	AGUCCAAAGUCUGAUCUGGTT	1022
PCNA	NM_002592	UUUCCUGUGCAAAAGACGGTT	1023
				PDGFRB	NM_002609	AAAGAAGUACCAGCAGGUGTT	1024
PDGFRB	NM_002609	UCCAUCCACCAGAGUCUAGTT	1025
				PDGFRB	NM_002609	UUUGCUGAGCUGCAUCGGATT	1026
PDZGEF2	NM_016340	AACCCUCAUCCACAGGUGATT	1027
				PDZGEF2	NM_016340	CCGACUGAGUACAUCGAUGTT	1028
PDZGEF2	NM_016340	GCCAGAUUCGACUGAUUGUTT	1029
				PIK3C2A	NM_002645	AACGAGGAAUCCGACAUUCTT	1030
PIK3C2A	NM_002645	UGAUGAGCCCAUCCUUUCATT	1031
				PIK3C2A	NM_002645	UGCUUCAACGGAUGUAGCATT	1032
POLS	NM_006999	ACAGAGACGCCGAAAGUACTT	1033
				POLS	NM_006999	CUAGCGACAUAGACCUGGUTT	1034

POLS	NM_006999	GCAAAUGAAUUGGCCUGGCTT	1035
				PPARG	NM_015869	AAUGACAGACCUCAGACAGTT	1036
PPARG	NM_015869	UAAGCCUCAUGAAGAGCCUTT	1037
				PPARG	NM_015869	UGUCAGUACUGUCGGUUUCTT	1038
PRC1	NM_003981	AAGCAUAUCCGUCUGUCAGTT	1039
				PRC1	NM_003981	AGGCUUCCAAAUCUGAUGCTT	1040
PRC1	NM_003981	GGAACUCUUUGAAGGUGUCTT	1041
				PRKACA	NM_002730	GAAUGGGGUCAACGAUAUCTT	1042
PRKACA	NM_002730	GGACGAGACUUCCUCUUGATT	1043
				PRKACA	NM_002730	GUGUGGCAAGGAGUUUUCUTT	1044
PRKCB1	NM_002738	AGAGCAUGCAUUUUUCCGGTT	1045
				PRKCB1	NM_002738	GGAGCCCCAUGCUGUAUUUTT	1046
PRKCB1	NM_002738	UUGGAUGUUAGCGGUACUCTT	1047
				PRKCL1	NM_002741	CACAAGAUCGUCUACAGGGTT	1048
PRKCL1	NM_002741	CACCAGUGAAGUCAGCACUTT	1049
				PRKCL1	NM_002741	GAUUUCAAGUUCCUGGCGGTT	1050
PRKCM	NM_002742	AAUGAAUGAGGAGGGUAGGTT	1051
				PRKCM	NM_002742	CCUUCAUCACCCUGGUGUUTT	1052
PRKCM	NM_002742	GUUCCCUGAAUGUGGUUUCTT	1053
				PRKWNK3	AJ409088	ACCAAGCAGCCAGCUAUACTT	1054
PRKWNK3	AJ409088	CUAAUGACAUCUGGGACCUTT	1055
				PRKWNK3	AJ409088	CUACGAAGGAAAACGUCAGTT	1056
PRKY	NM_002760	AGACAGUGAAGCUGGUUGUTT	1057
				PRKY	NM_002760	GAAUUUCUGAGGACGAGCUTT	1058
PRKY	NM_002760	UCAGAUUUGGGCCAGAGUUTT	1059
				PTEN	NM_000314	UGGAGGGGAAUGCUCAGAATT	1060
PTEN	NM_000314	AAGGCAGCUAAAGGAAGUGTT	1061
				PTEN	NM_000314	UAAAGAUGGCACUUUCCCGTT	1062
PTK6	NM_005975	AACACCCUCUGCAAAGUUGTT	1063
				PTK6	NM_005975	CCGUGGUUCUUUGGCUGCATT	1064
PTK6	NM_005975	UCAGGCUUAUCCGAUGUGCTT	1065
				PTTG1	NM_004219	AACAGCCAAGCUUUUCUGCTT	1066
PTTG1	NM_004219	GGCUUUGGGAACUGUCAACTT	1067
				PTTG1	NM_004219	UCUGUUGCAGUCUCCUUCATT	1068
RALA	NM_005402	AGACAGGUUUCUGUAGAAGTT	1069
				RALA	NM_005402	GUCCAGAUCGAUAUCUUAGTT	1070
RALA	NM_005402	GUUUAGCCAAGAGAAUCAGTT	1071
				RALBP1	NM_006788	AAUGAAGAGGUCCAAGGGATT	1072
RALBP1	NM_006788	AGGACCCGUGCAUCUUACUTT	1073
				RALBP1	NM_006788	GCUAAAAGACAGGAGUGUGTT	1074
RAP1A	NM_002884	CAGUGUAUGCUCGAAAUCCTT	1075
				RAP1A	NM_002884	GAUGAGCGAGUAGUUGGCATT	1076
RAP1A	NM_002884	UUGGAAAGUGCCAGCAUUCTT	1077
				RASA2	NM_006506	AACUGAUGACCUGGGGUCUTT	1078
RASA2	NM_006506	CAAGCAGAGAGCUCACCUATT	1079
				RASA2	NM_006506	GAAAACAAGCAAUCCGCAGTT	1080
RET	NM_000323	CUUCGCAGAAAAGAGUCGGTT	1081
				RET	NM_000323	GACAUCCAGGAUCCACUGUTT	1082
RET	NM_000323	GUGUGCCGAACUUCACUACTT	1083
				RHOK	NM_002929	AGUACACAGCAGGUUCAUCTT	1084
RHOK	NM_002929	CGUGAAUGAGGAGAACCCUTT	1085
				RHOK	NM_002929	GUUUAAGGAGGGGCCUGUGTT	1086

RPS6KA6	NM_014496	CCUCCUUUCAAACCUGCUUTT	1087
				RPS6KA6	NM_014496	GAGGUUCUGUUUACAGAGGTT	1088
RPS6KA6	NM_014496	UCAGCCAGUGCAGAUUCAATT	1089
				SGK2	NM_016276	AGAGCCUUAUGAUCGAGCATT	1090
SGK2	NM_016276	CUCUAUCAUGCCUGCUCCUTT	1091
				SGK2	NM_016276	GAGAAGGACCUGUGAAACUTT	1092
SKP2	NM_005983	AAGAACCAGGAGAUAUGGGTT	1093
				SKP2	NM_005983	GGUCUCUGGUGUUUGUAAGTT	1094
SKP2	NM_005983	UUUGCCCUGCAGACUUUGCTT	1095
				SRC	NM_005417	GAACCGGAUGCAGUUGAGCTT	1096
SRC	NM_005417	GCCGGAAUACAAGAACGGGTT	1097
				SRC	NM_005417	GUGGCUCUUAUCCGCAUGATT	1098
SRPK1	NM_003137	CGCUUAUGGAACGUGAUACTT	1099
				SRPK1	NM_003137	GCAACAGAAUGGCAGCGAUTT	1100
SRPK1	NM_003137	GUUCUAAUCGGAUCUGGCUTT	1101
				STAT3	NM_139276	AUGCCACAGGCCACCUAUATT	1102
STAT3	NM_139276	CGACCUGCAGCAAUACCAUTT	1103
				STAT3	NM_139276	GAAUCACAUGCCACUUUGGTT	1104
STAT4	NM_003151	ACACAGAUCUGCCUCUAUGTT	1105
				STAT4	NM_003151	CCCUACAAUAAAGGCCGGUTT	1106
STAT4	NM_003151	UUAGGAAGGUCCUUCAGGGTT	1107
				STAT5A	NM_003152	CCUGUGGAACCUGAAACCATT	1108
STAT5A	NM_003152	GUCUAUGAUGCUGUUGCCCTT	1109
				STAT5A	NM_003152	UGAGAUGAUUCAGAAGGGGTT	1110
STK4	NM_006282	CACCAUUUUGGAUGGCUCCTT	1111
				STK4	NM_006282	GGAAAACCAGAUCAACAGCTT	1112
STK4	NM_006282	UUCUGGAUGGCUUGCCUCATT	1113
				STK6	NM_003600	ACAGUCUUAGGAAUCGUGCTT	1114
STK6	NM_003600	GCACAAAAGCUUGUCUCCATT	1115
				STK6	NM_003600	UUGCAGAUUUUGGGUGGUCTT	1116
TCF3	M31523	AAAGACCCCGUGUAAACCUTT	1117
				TCF3	M31523	ACCUCAAGGCCAGCUCAAUTT	1118
TCF3	M31523	AUGGGGCAUUUUGUUGGGATT	1119
				TERT	NM_003219	CACCAAGAAGUUCAUCUCCTT	1120
TERT	NM_003219	GAGUGUCUGGAGCAAGUUGTT	1121
				TERT	NM_003219	GUUUGGAAGAACCCCACAUTT	1122
TGFBR1	NM_004612	GACAUGAUUCAGCCACAGATT	1123
				TGFBR1	NM_004612	UUCCUCGAGAUAGGCCGUUTT	1124
TGFBR1	NM_004612	UUUGGGAGGUCAGUUGUUCTT	1125
				TK2	NM_004614	GAUGCCAGAAGUGGACUAUTT	1126
TK2	NM_004614	UACCUGGAAGCAAUUCACCTT	1127
				TK2	NM_004614	UUAUGCUGCAUUUGGCUGGTT	1128
top2B	NM_001068	ACAUUCCCUGGAGUGUACATT	1129
				top2B	NM_001068	GAGGAUUUAGCGGCAUUUGTT	1130
top2B	NM_001068	GCUGCUGGACUGCAUAAAGTT	1131
				top3A	NM_004618	GAUCCUCCCUGUCUAUGAGTT	1132
top3A	NM_004618	GCAAAGAAAUUGGACGAGGTT	1133
				top3A	NM_004618	GGCGAAAACAUCGGGUUUGTT	1134
top3B	NM_003935	CAAAUGGGACAAAGUGGACTT	1135
				top3B	NM_003935	CUUUGACCUGAAGGGCUCUTT	1136
top3B	NM_003935	UCCAGUCCUUCAAACCAGATT	1137
				WASL	NM_003941	AAACAGGAGGUGUUGAAGCTT	1138

WASL	NM_003941	AAGUGGAGCAGAACAGUCGTT	1139
				WASL	NM_003941	GGACAAUCCACAGAGAUCUTT	1140
WEE1	NM_003390	AUCGGCUCUGGAGAAUUUGTT	1141
				WEE1	NM_003390	CAAGGAUCUCCAGUCCACATT	1142
WEE1	NM_003390	UGUACCUGUGUGUCCAUCUTT	1143
				WISP1	NM_003882	AAAUGCCUGUCUCUAGCUGTT	1144
WISP1	NM_003882	AUGGCCAGUUUUCUGGUAGTT	1145
				WISP1	NM_003882	CCUGGGCAUUGUUGAGGUUTT	1146
WISP3	NM_003880	ACAGUUUUGUCACUGGCCCTT	1147
				WISP3	NM_003880	CAAAAUGGACUCCCUGCUCTT	1148
WISP3	NM_003880	CCAGGGGAAAUCUGCAAUGTT	1149
				WNT1	NM_005430	ACGGCGUUUAUCUUCGCUATT	1150
WNT1	NM_005430	CCCUCUUGCCAUCCUGAUGTT	1151
				WNT1	NM_005430	CUAUUUAUUGUGCUGGGUCTT	1152
WNT2	NM_003391	AUUUGCCCGCGCAUUUGUGTT	1153
				WNT2	NM_003391	AACGGGCGAUUAUCUCUGGTT	1154
WNT2	NM_003391	AGAAGAUGAAUGGUCUGGCTT	1155
				WT1	NM_024426	CACUGGCACACUGCUCUUATT	1156
WT1	NM_024426	GACAAGAUACCGGUGCUUCTT	1157
				WT1	NM_024426	GACACCAAAGGAGACAUACTT	1158
	NM_017719	AGACCUAAUCACACGGAUGTT	1159
					NM_017719	AGAUAGCGGGUUCACCUACTT	1160
	NM_017719	GUUGACAGACUUUGGGUUCTT	1161
					XM_168069	ACUCCAUCUGGUUGACCUGTT	1162
	XM_168069	GAUUCAGGUGGAACUGAACTT	1163
					XM_168069	GCACCAAGCUCCUCUGAUGTT	1164
	XM_170783	ACCGACACUUUGGCUUCCATT	1165
					XM_170783	GAUGAGCGCGGGAAUGUUGTT	1166
	XM_170783	UGGCCGAGGCCUUCAAGCUTT	1167
					XM_064050	CAUCAAUCACUCUCUGCUGTT	1168
	XM_064050	CUAACCCAGGAUGUUCAGGTT	1169
					XM_064050	GACACUCACCAUGCUGAAATT	1170
	XM_066649	AAGGGUGACUUUGUGUCCUTT	1171
					XM_066649	ACCAGGAACAAACCUGUUGTT	1172
	XM_066649	UUUGAAGGUGGCCCUCCUATT	1173
					XM_089006	AAAUCGAGAAGGAGGCUCATT	1174
	XM_089006	AUAGUGACCGUCCCUUUGATT	1175
					XM_089006	CCAGGUUCCUCCAAAGAUGTT	1176
	NM_145754	AAGGGUUCAGCAUCUGACUTT	1177
					NM_145754	CCUGGAGACAUUGCACCAGTT	1178
	NM_145754	GGUGCUACCUCCUUUCCAGTT	1179
					NM_017596	AGUUGCCCACCCUGUUUUUTT	1180
	NM_017596	GAAAGAAUCCGUCCGCAUGTT	1181
					NM_017596	GCAGCCAGAACUCUCAAAGTT	1182
	NM_139286	GUUGUAGGAGGCAGUGAUGTT	1183
					NM_139286	UCUCAAUUUGGAAGUCUUGTT	1184
	NM_139286	UGAUCAAUGAUCGGAUUGGTT	1185
					NM_014885	ACCAGGAUUUGGAGUGGAUTT	1186
	NM_014885	CAAGGCAUCCGUUAUAUCUTT	1187
					NM_014885	GUGGCUGGAUUCAUGUUCCTT	1188
	NM_016263	CCAGAUCCUUGUCUGGAAGTT	1189
					NM_016263	CGACAACAAGCUGCUGGUCTT	1190

	NM_016263	GAAGCUGUCCAUGUUGGAGTT	1191
					NM_013367	AGCCAGCAGAUGUAAUUGGTT	1192
	NM_013367	CAUUUCAAUGAGGCUCCAGTT	1193
					NM_013367	GUCAUUUACAGAGUGGCUCTT	1194
	NM_018492	AGGACACUUUGGGUACCAGTT	1195
					NM_018492	GACCCUAAAGAUCGUCCUUTT	1196
	NM_018492	GCUGAGGAGAAUAUGCCUCTT	1197
					XM_168069	CAAAGUUAUUAGCCCCAAGTT	1198
	XM_168069	CAGAGGCCAAGUAUAUCAATT	1199
					XM_168069	CCUGCAGAUUUGCACAGCGTT	1200
	NM_021170	AUCCUGGAGAUGACCGUGATT	1201
					NM_021170	GCCGGUCAUGGAGAAGCGGTT	1202
	NM_021170	UGGCCCUGAGACUGCAUCGTT	1203
					NM_019089	CCCCUCCAUGCUCAGAACUTT	1204
	NM_019089	CCUAUCUGGGAAGCCUGUGTT	1205
					NM_019089	UGCCCCAGUGACAAUAACATT	1206
	AK024504	AGAGAGCUGGACCAUUCAUTT	1207
					AK024504	AUGAGCAAUGCGGAUAGCUTT	1208
	AK024504	GCCAUGUGUCUGAUGACAUTT	1209
					AB002301	AGACAAAGAGGGGACCUUCTT	1210
	AB002301	GAAAGUCUAUCCGAAGGCUTT	1211
					AB002301	UGCCUCCCUGAAACUUCGATT	1212
	NM_018401	AGGUAUGCAUCGUGCAGAATT	1213
					NM_018401	GCAAUCAAACCGUCAUGACTT	1214
	NM_018401	UAUCCUGCUGGAUGAACACTT	1215
					NM_016457	CAUUGUCCACUGUGACUUGTT	1216
	NM_016457	UGAAGUGGCCAUUCUGCAGTT	1217
					NM_016457	UGUGGACAUUGCCACUGUCTT	1218
	NM_005200	AUGAUCGCACCGCAGAGGUTT	1219
					NM_005200	UACAUGACGUACUUGAGUGTT	1220
	NM_005200	UGCUAAGGGGAUCGGACAUTT	1221
					NM_024322	ACCACUCCGGAUACAUCACTT	1222
	NM_024322	ACUAAGGCGUCUGCGAGAUTT	1223
					NM_024322	GGACCUCACAGCAACUCUUTT	1224
	NM_017769	CUGGUUGCAGUUCCAUUCCTT	1225
					NM_017769	GUGAGCAUCCUGGAUCAAATT	1226
	NM_017769	UUCAGAGAGUCCACACACCTT	1227
					NM_019013	AAAACCCCCGGGAGUCGUCTT	1228
	NM_019013	AGUGGCACCAAGUGGCUGGTT	1229
					NM_019013	GAAACCUGCUUUGUCAUUUTT	1230
	AI338451	CUGAUGCACUUUGCUGCAGTT	1231
					AI338451	CUGCAGGUUCAAAUCCCAGTT	1232
	AI338451	GGGGAAAAAGCUUUGCGUUTT	1233
					NM_018123	UAUCGAGCCACCAUUUGUGTT	1234
	NM_018123	UGAUGCAUAUAGCCGCAACTT	1235
					NM_018123	UGCACAGGGCCAAAGUUGATT	1236

6.4. embodiment 4: as the BRCA1/BARD1E3 ubiquitin ligase enzyme of cancer therapy drug target

Embodiment 2 and 3 has described the siRNA screening of the gene of the cell killing of identifying that enhancing DNA disrupting agent causes.In the present embodiment, with or without cisplatin treated HeLa cell, and studied the sensitization (Figure 19) that a member by the BRCC mixture causes.BRCA1, BRCA2, BARD1 and RAD51 are the most tangible in the specific gene, it is responsive that the destruction of described specific gene makes cell destroy DNA, BRCA1, BRCA2, BARD1 and RAD51 strengthen member (Dong et al., the Mol Cell.2003Nov that DNA destroys the BRCC mixture of back cell survival; 12 (5): 1087-99).The sensitization that BRCA1, BRCA2 and BARD1 cause all is a dose-dependently at cis-platinum concentration, but has only observed the sensitization that RAD51 causes (Fig. 1) when hanging down cis-platinum concentration.In other experiment, find that the destruction of BRCA1 and BRCA2 makes cis-platinum reduce＞about 4 times (data not shown goes out) the IC50 concentration of the inhibition of HeLa cell growth.The silence that BRCA1, BRCA2 and BARD1siRNA set cause is about 85%-98% (data not shown goes out).Table IV has been listed the BARD1 that uses in the present embodiment and the siRNA sequence of RAD51.

The remarkable part of these discoveries is the product and holoenzyme mixture (BRCC) association (Dong et al., the Mol Cell.2003Nov that strengthen the cell survival after DNA destroys of BRCA1, BRCA2, BARD21 and RAD51 gene; 12 (5): 1087-99).This mixture has E3Ub ligase enzyme activity, and great majority wherein can be used as the BRCAl/BARD1 heterodimer and reclaim (Dong et al., Mol Cell.2003Nov; 12 (5): 1087-99; Brzovicet al., Nat Struct Biol.2001Oct; 8 (10): 833-7).These find to show strongly that BRCC participates in the susceptibility of mediation to cis-platinum in our siRNA screening.Unexpectedly, the member's (FANCA, FANCC, FANCE and FANCF) of another kind of many subunits mixture-FANC mixture siRNA set participates in resistance (Taniguchiet al., the Nat Med.2003May of decision to cis-platinum; 9 (5): 568-74), it does not increase susceptibility (data not shown goes out) in our mensuration.

In order to determine that BRCA1 or BRCA2 destroy the sensitization to cis-platinum that causes and whether be subjected to that TP53 expresses the influence that exists or lack in the target cell, used by the stably express target and decide the TP53 positive of the coupling that the short hairpin RNA (shRNA) of TP53 produces and negative cells (referring to embodiment 2).Dye TP53 positive or negative cell with the siRNA of BRCA1 or BRCA2 set excess revolutions, use cisplatin treated, and with Alamar Blue analysis of cells grow (Figure 20).When with BRCA1 or BRCA2siRNAs (the about 0.1nM of IC50) transfection, sensitivity was about 10 times when the TP53 negative cells was used contrast siRNA (luciferase, the about 1nM of IC50) transfection to the cis-platinum ratio.In lower cis-platinum concentration, BRCA1 or BRCA2 destroy the back to the sensibilized of cis-platinum even more remarkable.After BRCA1 or BRCA2 destroyed, the TP53 positive cell was to cis-platinum more insensitive (the about 0.4nM of IC50).In this is analyzed, BRCA1 or BRCA2 destroy the back to the sensibilized of cis-platinum on the order of magnitude with sensibilized similar (data not shown goes out) after CHEK1 destruction.Also can use cell cycle analysis, research BRCA1 and BRCA2 destroy the sensibilized of back to the DNA disrupting agent.The TP53 positive and negative cells are dyed in siRNA set excess revolutions with BRCA1 or BRCA2, with a kind of processing in some DNA disrupting agents (cis-platinum, camptothecine, Zorubicin and bleomycin), and by flow cytometry cell cycle destruction.In all cases, the TP53 negative cells BRCA1 and BRCA2 destroy the back than in the luciferase cells transfected to DNA disrupting agent more responsive (data not shown goes out).These cells were shown in Figure 21 to the reaction of bleomycin after BRCA1 destroyed.BRCA1 destroys after handling the TP53 negative cells with bleomycin, has caused more inferior G1 cell (dead cell) than handling the TP53 positive cell.Described result shows that the cell that lacks TP53 destroys the back at BRCA1 and destroys more responsive to DNA.Figure 22 represents to prove RAD51/ Zorubicin synergy stronger result in the TP53-cell.

The clone of using in the present embodiment is positive A549 cell of HeLa cell, TP53 and the negative A549 cell of TP53.Decide the short hairpin RNA (shRNA) of TP53 by the stable transfection target, prepared the positive and negative cells of the TP53 of coupling to (monthly highlthighlight, Nov.2003).With the siRNA of 100nM (every kind of siRNA 33nM) set (set of three kinds of siRNA of each gene) transfectional cell, or with the single siRNA transfectional cell of 100nM.Following siRNA:Luc contrast, BRCA1, BRCA2 and BARD1 set have been used in our research.Handle cells transfected with the DNA disrupting agent of various concentration then.The concentration of the every kind of reagent that uses in the cell cycle analysis is as follows: for the HeLa cell, use Zorubicin (10nM), camptothecine (6nM), cis-platinum (400ng/ml), ametycin (40nM), bleomycin (100ng/ml); For other clone, use Zorubicin (200nM), camptothecine (200nM), cis-platinum (2ug/ml) ametycin (400nM), bleomycin (5ug/ml).

Be performed as follows the siRNA transfection: in transfection the day before yesterday, the clone that 2000 (or 100) microlitre is selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) grow to the about 90% cervical cancer HeLa cell (ATCC that converges in, Cat.No.CCL-2) be planted in the tissue culturing plate of 6 holes (or 96 holes) with 45,000 (or 2000) cells/well.For each transfection, with 70 microlitre OptiMEM (Invitrogen) with (Dharmacon, Lafayette CO) mix from 5 microlitre siRNA of 20 micromole's liquid storages.For each transfection, 20 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 25 microlitre OptiMEM/Oligofectamine mixtures are mixed with 75 microlitre OptiMEM/siRNA mixtures, and at room temperature incubation 15-20 minute.100 (or 10) microlitre transfection mixture is distributed in each hole of 6 holes (or 96 holes) plate, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.

After 4 hours, the DMEM/10% foetal calf serum that contains or do not contain the DNA disrupting agent in 100 microlitres/hole is added each hole, to reach the final concentration of every kind of reagent mentioned above.With plate incubation 68 hours again under the condition of 37 ℃ and 5%CO2.Analysis is from the cell cycle spectrum of the sample of 6 orifice plates, with the cell growth of Alamar Blue assay method analysis from the sample of 96 orifice plates.

For cell cycle analysis, will be from the supernatant liquor and the cytomixis of gathering in the crops by tryptic digestion in each hole.Then with 1200rpm with centrifugal 5 minutes of mixture.With 70% ice-cold ethanol with about 30 minutes of cell fixation.With PBS once, be suspended in 0.5ml again and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), and 37 ℃ of following incubations 30 minutes with the fixed cell washing.(Becton Dickinson) carries out flow cytometry with the FACSCalibur flow cytometer, and with FlowJo software (TreeStar, Inc) analytical data.Measure necrocytosis with inferior G1 cell mass.If (siRNA+DMSO) summation of the inferior G1 cell mass of sample and (Luc+ medicine) sample is greater than the inferior G1 cell mass of (siRNA+ medicine) sample, we determine that the siRNA silence is to the sensitization of DNA destructive.

Analyze for Alamar Blue, remove the substratum of 96 orifice plates, add the 100uL/ hole contain 10% (vol/vol) alamar Blue reagent (BioSource International, Inc) and the perfect medium of 1 percent volume 1M Hepes damping fluid tissue culture reagent.Then with plate at 37 ℃ of following incubation 1-4 hours, by exciting and detect emission at 590nm and measure fluorescence at 544nm with SPECTRAMax Gemini-Xs spectrofluorimeter (Molceular Devices).Background (acellular) is proofreaied and correct fluorescent signal.Cell response (survival) under the DNA disrupting agent exists is measured as the per-cent of the control cells growth that does not exist under the DNA disrupting agent condition.

BRCA1 has a lot of functions, but unique known enzyme function is an E3Ub ligase enzyme activity.This activity strengthens by the association of BARD1 and BRCA1, and causes the BRCA1/BARD1 mixture by unconventional ubiquitin K6 key and self ubiquitination (Wu-Baer et al., J Biol Chem.2003Sep 12; 278 (37): 34743-6; Chen et al., J Biol Chem.2002Jun 14; 277 (24): 22085-92).Obtainable evidence shows that BRCA1 E3 Ub ligase enzyme activity is that its DNA repairing effect is necessary.Its Ub ligase enzyme activity has been eliminated in the sudden change of tending to cancer in the BRCA1 RING structural domain, and (Ruffner et al., Proc Natl Acad SciU S A.2001Apr 24 to gamma-ray hypersensitivity for the mankind mastopathy cell that these sudden changes can not reverse no BRCA1; 98 (9): 5134-9).In addition, the BRCA1 of siRNA mediation destroys, blocked in the nucleus as accept that DNA in the gamma-rays radiating cell repairs and the nucleus position in activation site, the outpost of the tax office in multipass at deposition (Morris et al., the Hum Mol Genet.2004Apr15 of protein structure; 13 (8): 807-17).The ubiquitin key (K6) that is important to note that the BRCA1 mediation is different from ubiquitin key (K48) (Wu-Baer etal., the J Biol Chem.2003Sep 12 that makes albumen be subjected to the proteasome degraded; 278 (37): 34743-6; Morris et al., HumMol Genet.2004Apr 15; 13 (8): 807-17).Also do not know at present the function of K6 key, but known it can have signal transfer function.

In sum, discovery prompting in these discoveries and the document, the active inhibitor of BRCA1 E3 Ub ligase enzyme may be an effective anticancer agent, because with respect to normal cell (the TP53 positive), it can strengthen the treatment window of DNA disrupting agent to tumour cell (great majority are TP53 feminine genders).Carried out the BRCA1 level to cis-platinum susceptibility enhanced dose-dependently and multipass in the study on deposition of albumen in the nucleus position, whether have cause-effect relationship to observe these incidents.Also studied the chemical inhibitor of BRCA1 E3 Ub ligase enzyme, to establish the effect of multiubiquitination in the DNA destructive is repaired.

Prompting exists other to destroy the evidence of the E3 Ub ligase enzyme that works in the reparation from yeast research (Spence et al., Mol Cell Biol.1995Mar at DNA; 15 (3): 1265-73), show that DNA destroys reparation and need have the specific Ub ligase enzyme of non-proteolytic (K63 key).In order to promote to participate in the evaluation that DNA destroys the ligase enzyme of repairing, we have added the siRNA that has a plurality of E3 ligase enzymes of analog structure domain structure (nameless structural domain ligase enzyme) with BRCA1 in our siRNA library, wish to find to make cell that DNA is destroyed responsive those by our library screening.

The siRNA sequence of Table IV BARD1 and RAD51

	Adopted sequence is arranged	Serial ID	The gene title	SEQ?ID?NO
					5093	CAGUAAUUCUUAAGGCUAATT	NM_000465	BARD1	1237
5094	CUCCUGAGAAGGUCUGCAATT	NM_000465	BARD1	1238
					5095	CGCAGAAGCAGGCUCAACATT	NM_000465	BARD1	1239
6920	GUUAGAGCAGUGUGGCAUATT	NM_002875	RAD51	1240
					6921	GGUAUGCACUGCUUAUUGUTT	NM_002875	RAD51	1241
6922	CAGAUUGUAUCUGAGGAAATT	NM_002875	RAD51	1242

7. the reference of quoting

All reference of quoting in this is incorporated herein in full for all purposes, its degree is as specific and point out to be introduced as all purposes separately and introduce each independent open source literature or patent or patent application in full.

Can carry out many modifications and variations to the present invention, and not leave its spirit and scope, this it will be apparent to those skilled in the art that.Embodiment described herein only is to illustrate for example, and the present invention only is subjected to the qualification of whole equivalency range of claims and described claim.

Claims (218)

1. the method for identified gene, the product of described gene is regulated the effect of a kind of reagent to a kind of cell of cell type, and described method comprises

(a) a large amount of group of one or more cells of described cell type is contacted with described reagent, wherein the group of each described one or more cell comprises one or more the different siRNAs (siRNA) from a large amount of different siRNA, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target;

(b) effect to the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; With

(c) effect of the cell of the described cell type of arbitrary described heterogeneic siRNA is different calmly to not comprising target if described reagent is to the effect of the group of described one or more cells of described one or more different siRNA of comprising the fixed described gene of target and described reagent, and then identified gene is that described its product is regulated the gene of described reagent to the effect of the cell of described cell type.

2. the process of claim 1 wherein and comprise one or more each described groups of cells among described a large amount of siRNA by before described contact procedure, obtaining with described one or more siRNA transfections.

3. the method for identified gene, the product of described gene is regulated the effect of a kind of reagent to a kind of cell of cell type, and described method comprises

(a) with comprising from a large amount of group of one or more cells of the described cell type of composition transfection of one or more different siRNAs (siRNA) of a large amount of different siRNA each, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target;

(b) described a large amount of group of one or more cells is contacted with described reagent;

(c) effect to the cell of the described cell type of the fixed arbitrary described heterogeneic siRNA transfection of target of no use compares to the effect of the group of each described one or more cell and described reagent with described reagent; With

(d) effect of the cell of the described cell type of arbitrary described heterogeneic siRNA is different calmly to not comprising target if described reagent is to the effect of the group of described one or more cells of described one or more different siRNA of comprising the fixed described gene of target and described reagent, and then identified gene is that described its product is regulated the gene of described reagent to the effect of the cell of described cell type.

4. any one method of claim 1-3, wherein with described reagent the effect of the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target is compared, described reagent strengthens the effect of the group of each described one or more cell of comprising described siRNA.

5. any one method of claim 1-3, wherein with described reagent the effect of the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target is compared, described reagent weakens the effect of the group of each described one or more cell of comprising described siRNA.

6. any one method of claim 1-3, wherein said reagent act on by gene or its encoded protein beyond the fixed arbitrary described different genes of described a large amount of siRNA targets.

7. the method for claim 3, wherein said a large amount of siRNA comprise the different siRNA of the kind of k at least of at least a gene in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.

8. the method for claim 7, its described one or more different siRNA of described at least a gene that hit surely comprise 2,3,4,5,6 or 10 kind of different siRNA.

9. the method for claim 7, wherein said a large amount of siRNA comprise each the different siRNA of the kind of k at least at least two kinds of different genes in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.

10. the method for claim 9, described one or more different siRNA of each in its described surely at least two kinds of different genes that hit comprise 2,3,4,5,6 or 10 kind of different siRNA.

11. the method for claim 9, wherein said a large amount of siRNA comprise each the different siRNA of the kind of k at least in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.

12. the method for claim 11, described one or more different siRNA of each in its described surely different genes that hits comprise 2,3,4,5,6 or 10 kind of different siRNA.

13. the method for claim 5, wherein said cell type are the cancer cells types.

14. the method for claim 13, wherein said cell type are the cancer cells types, and wherein said effect is the growth-inhibiting effect.

15. the method for claim 12, wherein said reagent are the KSP inhibitor.

16. any one method of claim 7-15, wherein said multiple different gene comprises the different gene of N kind at least, wherein N be selected from 5,10,100,1000 with 5000 kinds of different genes.

17. any one method of claim 1-3, wherein said different gene is different native gene.

18. the method for the gene of the primary target gene interaction in the cell of evaluation and a kind of cell type, described method comprises

(a) a large amount of group of one or more cells of described cell type is contacted with a kind of reagent, wherein said reagent is regulated described primary target expression of gene and/or by the proteic activity of described primary target genes encoding, and the group of wherein said cell comprises one or more the different siRNA among a large amount of different siRNA, described one or more different siRNA target phasings gene together, and described a large amount of different siRNA comprise the siRNA of the different secondary genes in the fixed described cell of difference target;

(b) effect to the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; With

(c) effect of the cell of the described cell type of the siRNA of arbitrary described different secondary genes is different calmly to not comprising target if described reagent is to the effect of the group of described one or more cells of one or more siRNA of comprising the fixed described gene of target and described reagent, and then identified gene is the gene of the described primary target gene interaction in the cell of described and described cell type.

19. the method for claim 18 wherein comprises one or more each described groups of cells among described a large amount of siRNA by obtaining with described one or more siRNA transfections before described contact procedure.

20. the method for the gene of the primary target gene interaction in the cell of evaluation and a kind of cell type, described method comprises

(a) with comprising from a large amount of group of one or more cells of the described cell type of composition transfection of one or more different siRNAs (siRNA) of a large amount of different siRNA each, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target;

(b) described a large amount of group of one or more cells of described cell type is contacted with a kind of reagent, wherein said reagent is regulated described primary target expression of gene and/or by the proteic activity of described primary target genes encoding;

(c) effect to the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; With

(d) effect of the cell of the described cell type of the siRNA of arbitrary described different secondary genes is different calmly to not comprising target if described reagent is to the effect of the group of described one or more cells of one or more siRNA of comprising the fixed described gene of target and described reagent, and then identified gene is the gene of the described primary target gene interaction in the cell of described and described cell type.

Decide described primary target gene and make its reticent siRNA 21. any one method of claim 18-20, wherein said reagent are targets.

22. any one method of claim 18-20, described reagent are the inhibitor of described primary target gene.

23. any one method of claim 18-20, wherein with described reagent the effect of the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target is compared, described reagent strengthens the effect of the group of described one or more cells of comprising described one or more siRNA.

24. any one method of claim 18-20, wherein with described reagent the effect of the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target is compared, described reagent weakens the effect of the group of described one or more cells of comprising described one or more siRNA.

25. the method for claim 20, wherein said a large amount of siRNA comprise the different siRNA of the kind of k at least of the fixed secondary gene of at least a described difference of target, and wherein said k is selected from 2,3,4,5,6 and 10.

26. the method for claim 25, its described one or more different siRNA of described at least a gene that hit surely comprise 2,3,4,5,6 or 10 kind of different siRNA.

27. the method for claim 18, wherein said a large amount of siRNA comprise each the different siRNA of the kind of k at least at least two kinds of different genes in the fixed secondary gene of described difference of target, and wherein said k is selected from 2,3,4,5,6 and 10.

28. the method for claim 27, described one or more different siRNA of each in its described surely at least two kinds of different genes that hit comprise 2,3,4,5,6 or 10 kind of different siRNA.

29. the method for claim 27, wherein said a large amount of siRNA comprise each the different siRNA of the kind of k at least in the fixed secondary gene of described difference of target, and wherein said k is selected from 2,3,4,5,6 and 10.

30. the method for claim 29, described one or more different siRNA of each in its described surely different genes that hits comprise 2,3,4,5,6 or 10 kind of different siRNA.

31. the method for claim 22, wherein said primary target gene is KSP.

32. the method for claim 18, wherein said multiple different gene comprises the different gene of N kind at least, wherein N be selected from 5,10,100,1000 with 5000 kinds of different genes.

33. any one method of claim 18-20, wherein said different secondary gene is different native gene.

34. any one method of claim 18-20, wherein said cell type are the cancer cells types.

35. the method for claim 8 or 26, the total siRNA concentration of one or more siRNA described in the wherein said composition is the optimal concentration that is used to make described target gene silence, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.

36. the method for claim 35, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

37. the method for claim 35, the concentration of each of wherein said one or more siRNA is approximately identical.

38. the method for claim 35, wherein said one or more siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.

39. the method for claim 35 does not have the concentration of a kind of siRNA to account for more than 80% of total siRNA concentration of described one or more siRNA, more than 50% or more than 20% in the wherein said composition.

40. the method for claim 35, the concentration of at least a siRNA accounts for more than 20% or more than 50% of total siRNA concentration of described one or more siRNA in the wherein said composition.

41. the method for claim 8 or 26, wherein select the concentration of each siRNA in the number of different siRNA and the described composition, make described composition cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

42. treatment suffers from the mammiferous method of cancer, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, wherein said Mammals carries out a kind of treatment, and described treatment comprises the KSP inhibitor to described administration treatment q.s.

43. treatment suffers from the mammiferous method of cancer, comprise to described administration i) treatment q.s a kind of reagent, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding with ii) treat the KSP inhibitor of q.s.

44. the method for claim 42 or 43, wherein said reagent reduce STK6 or TPX2 expression of gene described in the cell of described cancer.

45. the method for claim 42 or 43, wherein said reagent comprise the siRNA of fixed described STK6 of target or TPX2 gene.

46. the method for claim 45, wherein said reagent comprise 2,3,4,5,6 or the different siRNA of fixed described STK6 of 10 kind of target or TPX2 gene.

47. the method for claim 46, the total siRNA concentration of different siRNA described in the wherein said reagent is the optimal concentration that is used to make described STK6 or TPX2 gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.

48. the method for claim 47, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

49. the method for claim 47, wherein the concentration of each described different siRNA is approximately identical.

50. the method for claim 47, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.

51. the method for claim 47 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.

52. the method for claim 47, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said reagent.

53. the method for claim 47 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

54. the method for claim 45, wherein said Mammals is the people, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.

55. treatment suffers from the mammiferous method of cancer, comprise to described administration i) treatment q.s first kind of reagent, described first kind of reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, ii) treat second kind of reagent of q.s, described second kind of reagent is regulated the KSP expression of gene and/or by the proteic activity of described KSP genes encoding.

56. the method for claim 55, wherein said first kind of reagent comprise the siRNA of fixed described TPX2 of target or TPX2 gene, and described second kind of reagent comprises the siRNA of the fixed described KSP gene of target.

57. the method for claim 56, wherein said first kind of reagent comprise 2,3,4,5,6 or the different siRNA of fixed described STK6 of 10 kind of target or TPX2 gene.

58. the method for claim 57, total siRNA concentration of different siRNA is the optimal concentration that is used to make described STK6 or TPX2 gene silencing described in wherein said first kind of reagent, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.

59. the method for claim 58, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

60. the method for claim 58, wherein the concentration of each described different siRNA is approximately identical.

61. the method for claim 58, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.

62. the method for claim 58 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in wherein said first kind of reagent.

63. the method for claim 58, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in wherein said first kind of reagent.

64. the method for claim 58, wherein select the concentration of each siRNA in the number of different siRNA and the described first kind of reagent, make described first kind of reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

65. the method for claim 56, wherein said Mammals is the people, and the described siRNA of its described surely STK6 gene that hits is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.

66. the method for claim 45 or 56, wherein said Mammals is the people, and the described siRNA of its described surely TPX2 gene that hits is selected from the siRNA shown in SEQ ID NO:1237, SEQ IDNO:1238 and the SEQ ID NO:1239.

67. the assessment cell is to the method for the Growth Inhibition resistance of KSP inhibitor, described method comprises STK6 or the TPX2 expression of gene level of determining in the described cell, the described expression level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.

68. the method for claim 67, wherein by comprising the method for measuring described STK6 or TPX2 expression of gene level with one or more nucleotide probes, determine described STK6 or TPX2 expression of gene level, each in described one or more polynucleotide probes all comprises the nucleotide sequence in described STK6 or the TPX2 gene.

69. the method for claim 67 or 68, wherein said one or more polynucleotide probes are the polynucleotide probes on microarray.

70. the assessment cell is to the method for the Growth Inhibition resistance of KSP inhibitor, described method comprises the proteic abundance level of determining in the described cell by STK6 or TPX2 genes encoding, the described proteic described abundance level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.

71. the assessment cell is to the method for the Growth Inhibition resistance of KSP inhibitor, described method comprises the proteic activity level of determining in the described cell by STK6 or TPX2 genes encoding, the described activity level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.

72. the method for claim 70 or 71, wherein said cell are people's cells.

73. regulate the method for cell to the Growth Inhibition resistance of KSP inhibitor, comprise a kind of reagent that makes described cells contacting q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding.

74. cell is to the method for the Growth Inhibition resistance of KSP inhibitor in the adjusting Mammals, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding.

75. regulate the method for cell growth, comprise making described cells contacting i) a kind of reagent of q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding and the ii) KSP inhibitor of q.s.

76. claim 73,74 or 75 method, wherein said reagent reduces STK6 described in the described cell or TPX2 expression of gene.

77. claim 73,74 or 75 method, wherein said reagent comprise the siRNA of the fixed described STK6 gene of target.

78. the method for claim 77, wherein said reagent comprise 2,3,4,5,6 or the different siRNA of fixed described STK6 of 10 kind of target or TPX2 gene.

79. the method for claim 78, the total siRNA concentration of different siRNA described in the wherein said reagent is the optimal concentration that is used to make described STK6 or TPX2 gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.

80. the method for claim 79, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

81. the method for claim 79, wherein the concentration of each described different siRNA is approximately identical.

82. the method for claim 79, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.

83. the method for claim 79 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.

84. the method for claim 79, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said reagent.

85. the method for claim 79 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

86. the method for claim 77, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.

87. the method for claim 77, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.

88. identify and to regulate the compositions and methods of cell to the Growth Inhibition resistance of KSP inhibitor, wherein said reagent can be regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, described method comprise described KSP inhibitor when relatively having described reagent to the cell inhibiting effect of expressing described STK6 or TPX2 gene when not having described reagent described KSP inhibitor to expressing the cell inhibiting effect of described STK6 or TPX2 gene, the described inhibiting difference of wherein said KSP inhibitor identifies that described reagent can regulate the Growth Inhibition resistance of described cell to the KSP inhibitor.

89. identify and can regulate the compositions and methods of cell that wherein said reagent can regulate STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, described method comprises to the Growth Inhibition resistance of KSP inhibitor:

(a) first kind of cell of expressing described STK6 or TPX2 gene contacted existing with described KSP inhibitor, and measure first kind of growth-inhibiting effect;

(b) second kind of cell of expressing described STK6 or TPX2 gene contacted with described KSP inhibitor, and measure second kind of growth-inhibiting effect; With

(c) compare in described step (a) and described first kind and second kind of restraining effect of measuring (b),

Difference between wherein said first kind and the second kind of restraining effect identifies that described reagent can regulate the Growth Inhibition resistance of cell to the KSP inhibitor.

90. the method for claim 88 or 89, wherein said reagent comprise the molecule that reduces described STK6 or TPX2 genetic expression.

91. the method for claim 88 or 89, wherein said reagent are the siRNA that target is decided described STK6 or TPX2 gene.

92. the method for claim 91, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.

93. the method for claim 91, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.

94. comprise the cell of one or more different siRNAs (siRNA), STK6 or TPX2 gene in the fixed described cell of described siRNA target.

95. the cell of claim 94, wherein said one or more different siRNA comprise 2,3,4,5,6 or 10 kind of different siRNA.

96. the cell of claim 95, wherein said cell is to produce by the composition transfection with described one or more different siRNA, total siRNA concentration of wherein said composition is the optimal concentration that is used to make described STK6 or TPX2 gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, do not increase reticent level substantially.

97. the cell of claim 96, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

98. the cell of claim 96, wherein the concentration of each described different siRNA is approximately identical.

99. the cell of claim 96, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.

100. the cell of claim 96, do not have in the wherein said composition concentration of a kind of siRNA account for more than 80% of total siRNA concentration of described different siRNA, more than 50% or more than 20%.

101. the cell of claim 96, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said composition.

102. the cell of claim 96, wherein select the concentration of each siRNA in the number of different siRNA and the described composition, make described composition cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

103. the cell of claim 94, wherein said cell are people's cells.

104. the cell of claim 103, wherein said cell is people's cell, and each of wherein said one or more different siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and the SEQ IDNO:6.

105. the cell of claim 103, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.

106. the cell of claim 94, wherein said cell are the mouse cells.

107. be used for the microarray of diagnosis cell to the Growth Inhibition resistance of KSP inhibitor, described microarray comprises one or more polynucleotide probes, wherein each described polynucleotide probes comprises the nucleotide sequence in STK6 or the TPX2 gene.

108. be used for the test kit of diagnosis cell to the Growth Inhibition resistance of KSP inhibitor, described test kit comprises one or more polynucleotide probes that place one or more containers, and wherein each described polynucleotide probes comprises the nucleotide sequence in STK6 or the TPX2 gene.

109. be used to screen a kind of test kit of reagent, described reagent is regulated the Growth Inhibition resistance of cell to the KSP inhibitor, described test kit comprises the cell of (i) claim 94 that places one or more containers; (ii) KSP inhibitor.

110. be used for the treatment of the mammiferous test kit of suffering from cancer, this test kit comprises the adjusting STK6 of (i) q.s that places one or more containers or TPX2 expression of gene and/or by the proteic active reagent of described STK6 or TPX2 genes encoding; (ii) KSP inhibitor.

111. any one method of claim 42-43,67,70-71,74-75 and 88-89, wherein said KSP inhibitor are (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine.

112. claim 1,2 or 3 method, wherein the component to each described one or more cell drives capable contact procedure (a) into.

113. claim 18,19 or 20 method, wherein the component to each described one or more cell drives capable contact procedure (a) into.

114. the test kit of claim 109 or 110, wherein said KSP inhibitor are (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine.

115. the method for the gene of the primary target gene interaction in the cell of evaluation and a kind of cell type, described method comprises

(a) make a kind of reagent of one or more cells contacting of described cell type, wherein said reagent is regulated the secondary target expression of gene and/or by the proteic activity of described secondary target genes encoding, and first siRNA (siRNA) of the fixed described primary target gene of wherein said one or more cell expressing targets;

(b) more described reagent is to the effect to the cell of the described cell type of not expressing a described siRNA of the effect of described one or more cells of described clone and described reagent; With

(c) if described reagent is different to the effect of the cell of the described cell type of not expressing a described siRNA to the effect of described one or more cells of expressing a described siRNA and described reagent, then identify described secondary target gene for the cell of described cell type in the gene of primary target gene interaction.

116. the method for the gene of the primary target gene interaction in the cell of evaluation and a kind of cell type, described method comprises

(a) clone of the cell of the described cell type of first siRNA (siRNA) of the fixed described primary target gene of generation expression target;

(b) make a kind of reagent of one or more cells contacting of described clone, wherein said reagent is regulated the secondary target expression of gene and/or by the proteic activity of described secondary target genes encoding;

(c) more described reagent is to the effect to the cell of the described cell type of not expressing a described siRNA of the effect of described one or more cells of described clone and described reagent; With

(d) if described reagent is different to the effect of the cell of the described cell type of not expressing a described siRNA to the effect of described one or more cells of expressing a described siRNA and described reagent, then identify described secondary target gene for the cell of described cell type in the gene of primary target gene interaction.

117. the method for claim 116, a wherein said siRNA is expressed by the nucleotide sequence that is incorporated in the described cellular genome.

118. the method for claim 116, wherein said reagent comprise the fixed described secondary target gene of target and make one or more the 2nd siRNA of its silence.

119. the method for claim 116, wherein said reagent are the inhibitor of described secondary target gene.

120. the method for claim 118 is wherein compared the effect of the cell of the described cell type of not expressing a described siRNA with described reagent, described reagent strengthens the effect of described one or more cells of expressing a described siRNA.

121. the method for claim 118 is wherein compared the effect of the cell of the described cell type of not expressing a described siRNA with described reagent, described reagent weakens the effect of described one or more cells of expressing a described siRNA.

122. the method for claim 120, wherein said one or more the 2nd siRNA comprise the different siRNA of k kind at least, and wherein said k is selected from 2,3,4,5,6 and 10.

123. the method for claim 122, total siRNA concentration of the siRNA that the k kind is different at least described in the wherein said reagent is the optimal concentration that is used to make described secondary target gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, do not increase reticent level substantially.

124. the method for claim 123, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

125. the method for claim 123, the concentration of each of the siRNA that the wherein said kind of k at least is different is approximately identical.

126. the method for claim 123, the different siRNA concentration separately difference each other of the wherein said kind of k at least are less than 50%, less than 20% or less than 10%.

127. the method for claim 123 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.

128. the method for claim 123, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of the different siRNA of the described kind of k at least in the wherein said reagent.

129. the method for claim 123 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

130. the method for claim 122, wherein said cell type are the cancer cells types, and wherein said primary target gene is p53.

131. the method for claim 130 further comprises step

(e) in a large amount of different secondary target genes each, repeating step (b)-(d).

132. the method for claim 131, wherein said a large amount of secondary target gene comprise the different genes of number that is selected from down group at least: 5,10,100,1000 with 5000 kinds of different genes.

133. the method for claim 132, wherein said effect are the change of the cell of described cell type to the susceptibility of medicine.

134. the method for claim 133, wherein said medicine are the DNA disrupting agents.

135. the method for claim 134, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.

136. the method for claim 135, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.

137. assess the reactive method of a kind of cell of cell type, comprise to pharmacological agent

(a) make the described medicine of one or more cells contacting of described cell type, wherein said one or more cell expressing targets are decided first siRNA (siRNA) of primary target gene, and wherein said one or more cells are accepted a kind of processing of composition, and described composition is regulated one or more secondary target expression of gene and/or respectively by the proteic activity of described one or more secondary target genes encodings;

(b) make the described medicine of one or more cells contacting of described cell type, wherein said one or more cells are not expressed the siRNA (siRNA) of the fixed described primary target gene of target, and wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active described reagent of described secondary target genes encoding; With

(c) the described medicine of relatively measuring in step (a) is to the effect to described one or more cells of the described medicine of the effect of described one or more cells and measurement in step (b), thereby assesses the reactivity of described cell to described pharmacological agent.

138. assess the reactive method of a kind of cell of cell type to pharmacological agent, described method comprises

(a) clone of cell of described subclass type that target is decided first siRNA (siRNA) of primary target gene is expressed in preparation;

(b) make the described clone's who expresses a described siRNA the described medicine of one or more cells contacting, wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active reagent of described secondary target genes encoding;

(c) make the described medicine of one or more cells contacting of the described cell type of the siRNA (siRNA) of not expressing the fixed described primary target gene of target, wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active described reagent of described secondary target genes encoding; With

(d) the described medicine of relatively measuring in step (b) is to the effect to described one or more cells of the described medicine of the effect of described one or more cells and measurement in step (c), thereby assesses the reactivity of described cell to described pharmacological agent.

139. the method for claim 137 or 138 is wherein compared the effect of the cell of the described cell type of not expressing a described siRNA with described medicine, described medicine strengthens the effect of one or more cells of expressing a described siRNA.

140. the method for claim 137 or 138 is wherein compared the effect of the cell of the described cell type of not expressing a described siRNA with described medicine, described medicine weakens the effect of one or more cells of expressing a described siRNA.

141. the method for claim 137 or 138, wherein said composition comprise one or more inhibitor of described one or more secondary target genes.

142. the method for claim 137 or 138, wherein said composition comprise fixed described one or more second target genes of target and make one or more the 2nd siRNA of its silence.

143. the method for claim 142, wherein said one or more the 2nd siRNA comprise the different siRNA of k kind at least, and wherein said k is selected from 2,3,4,5,6 and 10.

144. the method for claim 143, total siRNA concentration of the siRNA that the k kind is different at least described in the wherein said reagent is the optimal concentration that is used to make described secondary target gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, do not increase reticent level substantially.

145. the method for claim 144, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

146. the method for claim 144, the concentration of each of the siRNA that the wherein said kind of k at least is different is approximately identical.

147. the method for claim 144, the different siRNA concentration separately difference each other of the wherein said kind of k at least are less than 50%, less than 20% or less than 10%.

148. the method for claim 144 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.

149. the method for claim 144, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of the different siRNA of the described kind of k at least in the wherein said reagent.

150. the method for claim 144 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

151. the method for claim 137 or 138, wherein said cell type are the cancer cells types, and wherein said primary target gene is p53.

152. the method for claim 138 further comprises step

(e) in a large amount of different secondary target genes each, repeating step (b)-(d).

153. the method for claim 137 further comprises step

(e) in a large amount of different secondary target genes each, repeating step (a)-(b).

154. the method for claim 152 or 153, wherein said a large amount of secondary target gene comprise the different genes of number that is selected from down group at least: 5,10,100,1000 with 5000 kinds of different genes.

155. the method for claim 154, wherein said medicine are the DNA disrupting agents.

156. the method for claim 155, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.

157. the method for claim 156, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.

158. treatment suffers from the mammiferous method of cancer, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated a kind of expression of gene and/or by the proteic activity of described genes encoding, wherein said Mammals carries out a kind of treatment, and described treatment comprises the composition that comprises one or more DNA disrupting agents to described administration treatment q.s.

159. treatment suffers from the mammiferous method of cancer, comprise to described administration i) treatment q.s a kind of reagent, described reagent is regulated a kind of expression of gene and/or by the proteic activity of described genes encoding with ii) treat the composition that comprises one or more DNA disrupting agents of q.s.

160. the method for claim 158 or 159, wherein said reagent make the expression decreased of described gene in the cell of described cancer.

161. the method for claim 158 or 159, wherein said reagent strengthen the expression of described gene in the cell of described cancer.

162. the method for claim 161, wherein said one or more DNA disrupting agents are selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

163. the method for claim 161, wherein said one or more DNA disrupting agents are selected from Zorubicin, camptothecine and cis-platinum, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

164. the method for claim 163, wherein said reagent comprise the siRNA of the fixed described gene of target.

165. the assessment cell is to a kind of method of Growth Inhibition susceptibility of reagent, described method comprises each transcriptional level of one or more genes of determining in the described cell, wherein be lower than each described transcriptional level of each gene of predetermined threshold levels, show that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.

166. the method for claim 165, wherein said reagent is the DNA disrupting agent, it is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

167. the method for claim 165, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

168. any one method of claim 166-167, wherein said one or more genes comprise about at least 5 to about 50 kinds of different genes.

169. the method for claim 168, wherein each described transcriptional level is than low 1.5 times, 2 times or 3 times of described threshold levels.

170. any one method of claim 166-167, wherein by comprising the method for measuring described gene transcription level with one or more nucleotide probes, determine described gene transcription level, each in described one or more polynucleotide probes all comprises the nucleotide sequence in the described gene.

171. the method for claim 170, wherein said one or more polynucleotide probes are the polynucleotide probes on microarray.

172. be used to assess the method for cell to the Growth Inhibition susceptibility of DNA disrupting agent, described method comprises the proteic abundance level of determining in the described cell by a kind of genes encoding, wherein be lower than the described proteic described abundance level of predetermined threshold levels, show that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.

173. the assessment cell is to the method for the Growth Inhibition susceptibility of DNA disrupting agent, described method comprises the proteic activity level of determining in the described cell by described genes encoding, the described activity level that wherein is higher than predetermined threshold levels shows that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.

174. the method for claim 172 or 173, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

175. the method for claim 174, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

176. the method for claim 172 or 173, wherein said cell are people's cells.

177. regulate the method for cell to DNA destructive susceptibility, described method comprises makes described cell contact with a kind of reagent of q.s, and described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding.

178. the method for claim 177, wherein said DNA destroys and is caused by the DNA disrupting agent.

179. the method for claim 178, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.

180. the method for claim 179, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.

181. regulate the method for cell growth, comprise and make described cells contacting i) a kind of reagent of q.s, described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding; The ii) DNA disrupting agent of q.s.

182. the method for claim 177 or 181, wherein said reagent reduces expression of gene described in the described cell.

183. the method for claim 177 or 181, wherein said reagent comprise the siRNA of the fixed described gene of target.

184. the method for claim 183, wherein said reagent comprise 2,3,4,5,6 or 10 kind of different siRNA of the fixed described gene of target.

185. the method for claim 184, total siRNA concentration of different siRNA is the optimal concentration that is used to make described gene silencing described in the wherein said reagent, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.

186. the method for claim 185, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

187. the method for claim 185, wherein the concentration of each described different siRNA is approximately identical.

188. the method for claim 185, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.

189. the method for claim 185 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.

190. the method for claim 185, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said reagent.

191. the method for claim 185 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

192. identify and to regulate the compositions and methods of cell to the Growth Inhibition susceptibility of DNA disrupting agent, wherein said reagent can be regulated and be selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, the expression of gene of BARD1 and RAD51 and/or by the proteic activity of described genes encoding, this method comprise described DNA disrupting agent when relatively having described reagent to the cell inhibiting effect of expressing said gene when not having described reagent described DNA disrupting agent to the cell inhibiting effect of expressing said gene, the described inhibiting difference of wherein said DNA disrupting agent identifies that described reagent can regulate the Growth Inhibition susceptibility of described cell to the DNA disrupting agent.

193. identify and to regulate the compositions and methods of cell to the Growth Inhibition susceptibility of DNA disrupting agent, wherein said reagent can regulate the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding, described method comprises

(a) exist the cell that makes first kind of expressing said gene under the condition of described reagent to contact, and measuring first kind of growth-inhibiting effect with described DNA disrupting agent;

(b) cell of second kind of expressing said gene is contacted with described DNA disrupting agent, and measure second kind of growth-inhibiting effect;

(c) compare in described step (a) and described first kind and second kind of restraining effect of measuring (b),

Difference between wherein said first kind and the second kind of restraining effect identifies that described reagent can regulate the Growth Inhibition susceptibility of described cell to described DNA disrupting agent.

194. the method for claim 192 or 193, wherein said cell expressing target is decided the siRNA of primary target gene.

195. the method for claim 194, wherein said primary target gene is p53.

196. the method for claim 192 or 193, wherein said reagent comprises the molecule that reduces described genetic expression.

197. the method for claim 196, wherein said reagent comprise the siRNA of the fixed described gene of target.

198. the method for claim 197, wherein said reagent comprise 2,3,4,5,6 or 10 kind of different siRNA of the fixed described gene of target.

199. the method for claim 198, total siRNA concentration of different siRNA is the optimal concentration that is used to make described gene silencing described in the wherein said reagent, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.

200. the method for claim 199, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.

201. the method for claim 199, wherein the concentration of each described different siRNA is approximately identical.

202. the method for claim 199, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.

203. the method for claim 199 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.

204. the method for claim 199, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said reagent.

205. the method for claim 199 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.

206. comprise the cell of one or more different siRNAs (siRNA), the gene that is selected from EPHB3, WEE1, ELK1, BRCA1, BRCA2, BARD1 and RAD51 in the fixed described cell of described siRNA target.

207. the method for claim 206, wherein said one or more siRNA comprise 2,3,4,5,6 or 10 kind of different siRNA.

208. the method for claim 206, wherein said cell are people's cells.

209. the method for claim 208, wherein said cell are the mouse cells.

210. be used for the microarray of diagnosis cell to the Growth Inhibition susceptibility of DNA disrupting agent, described microarray comprises one or more polynucleotide probes, and wherein each described polynucleotide probes comprises the nucleotide sequence in one or more genes that are selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

211. be used for the test kit of diagnosis cell to the Growth Inhibition susceptibility of DNA disrupting agent, described test kit comprises one or more polynucleotide probes that place one or more containers, and wherein each described polynucleotide probes comprises the nucleotide sequence in the gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.

Regulate the test kit of cell to the reagent of the Growth Inhibition susceptibility of DNA disrupting agent 212. be used to screen, described test kit comprises any one cell of (i) the claim 206-211 that places one or more containers; (ii) described DNA disrupting agent.

213. be used for the treatment of the mammiferous test kit of suffering from cancer, described test kit comprises a kind of reagent of (i) q.s that places one or more containers, and described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding; (ii) DNA disrupting agent.

214. any one method of claim 192-193, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.

215. the method for claim 214, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.

216. the test kit of claim 212, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.

217. the test kit of claim 216, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.

218. claim 21,117,137 or 138 method are wherein controlled the reticent level of described primary target gene.

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